T cell epitopes of hcmv and uses of thereof

ABSTRACT

The present invention relates to relates to T cell epitope peptides, proteins, nucleic acids and cells for use in immunother-apeutic methods. In particular, the present invention relates to the immunotherapy of viral infection. The present invention specifically relates to virus-associated T-cell peptide epitopes, alone or in combination with other virus-associated peptides that can serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-viral immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

The present invention relates to relates to T cell epitope peptides,proteins, nucleic acids and cells for use in immunotherapeutic methods.In particular, the present invention relates to the immunotherapy ofviral infection. The present invention specifically relates tovirus-associated T-cell peptide epitopes, alone or in combination withother virus-associated peptides that can serve as active pharmaceuticalingredients of vaccine compositions that stimulate anti-viral immuneresponses, or to stimulate T cells ex vivo and transfer into patients.Peptides bound to molecules of the major histocompatibility complex(MHC), or peptides as such, can also be targets of antibodies, solubleT-cell receptors, and other binding molecules.

BACKGROUND OF THE INVENTION

In healthy individuals, immune control of persistent humancytomegalovirus (HCMV) infection is effectively mediated byvirus-specific CD4+ and CD8+ T-cells. However, identification of therepertoire of T-cell specificities for HCMV is hampered by the immenseprotein coding capacity of this betaherpes virus.

HCMV-associated pathologies are a common cause of post-transplantmorbidity and mortality. Identification of the physiological targets ofanti-HCMV T-cell responses will allow for improvements of currenttreatments of these complications, e.g. by adoptive T-cell transfer.Although HCMV-derived T-cell epitopes could already be identified, someof these are recognized infrequently. Furthermore, research has mainlyfocused on subsets of HLA alleles, leaving gaps in the HLA coverage.

Primary infection with human cytomegalovirus (HCMV) is followed bylifelong latency with recurrent cycles of endogenous reactivation. Theprevalence of HCMV in adults ranges from 40 to 90% and increases withage (1, 2).

While in the immunocompetent host infection is usually controlled by anHCMV-specific immune response, severe damage is common inimmunologically restricted populations. Congenital infection of thefetus has considerable consequences, involving the central nervoussystem with sensorineural hearing loss, mental retardation or evendeath. HCMV infection or reactivation is a major cause of morbidity andmortality in immunocompromised individuals such as AIDS patients ortransplant recipients, since CD8+ but also CD4+ T-cell immunity plays acritical role in preventing lethal infection (3-5). Further, it isthought that subclinical infections with HCMV are involved in a varietyof diseases, for example certain cancers, inflammatory, hypertensive,and pulmonary diseases (6-10).

Current HCMV treatments include antiviral drugs and attempts to exploitthe humoral and cellular immune responses. Furthermore, considerableeffort is made on the development of an HCMV vaccine. For allimmunological therapies deeper insights into potential target structuresare vitally important. Since with its almost 236 kbp longdouble-stranded DNA genome, HCMV has the largest genome among humanherpesviruses, the majority of studies on cytotoxic T-lymphocyteresponses have so far been restricted to a very limited selection ofHCMV antigens; most prominent among them are the immunodominant antigenspp65 and IE1 (11-15).

A number of studies have clearly demonstrated that the HCMV-specificT-cell response targets a much broader spectrum of HCMV antigens(16-18). To date, the identification of most HCMV-specific T-celltargets has been based on prediction methods (16, 19, 20) or the use ofoverlapping peptides (18). The approach of direct isolation of viralligands from infected target cells, successfully used for some viralinfections (21-24), has been cumbersome due to strict control of peptidepresentation by HCMV encoded HLA class I (HLA-I) immunoevasins (25-30).

Glycoproteins encoded by the US6 gene family are able to impair thestability and localization of HLA-I. The glycoproteins US2 and US11 bindHLA-I and mediate their reverse transport into the cytosol forsubsequent degradation by the proteasome (31-33). US6 prevents theassembly of the HLA-I/peptide complexes by inhibiting the transport ofpeptides into the endoplasmic reticulum by the transporter associatedwith antigen processing (TAP) (26, 34). The product of US3 formscomplexes with assembled J32-microglobulin-associated HLA-I heavychains, thereby blocking maturation and translocation of HLA-I moleculesto the cell surface (25).

Therefore, development of better therapies and prevention strategies inHCMV is of considerable importance. Other objects of the presentinvention will become apparent to the person of skill when studying thefollowing more detailed description of the invention.

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variantsequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acidexchange and bind to molecule(s) of the major histocompatibility complex(MHC) and/or induce T cells cross-reacting with said variant peptide,and a pharmaceutical acceptable salts thereof, wherein said peptide hasan overall length of between 8 and 30, preferably 9 and 30, amino acids.

Preferred is a peptide or variant according to the present invention,wherein said peptide consists or consists essentially of an amino acidsequence according to any of SEQ ID NO: 1 to SEQ ID NO: 100 oroptionally comprises an extension of one N- and/or C-terminal,preferably naturally occurring, amino acid.

More preferred is a peptide or variant according to the presentinvention, wherein the amino acid sequence is selected from SEQ ID NO: 1to 4, 24 to 29, 40, 41, 51 to 55, 67, 68, 80, 87 to 89, and 99 to 101.

Here, the inventors present a novel approach which employs HCMV deletionmutant viruses, lacking HLA class I immunoevasins, to allow directidentification of naturally presented HCMV-derived HLA ligands by massspectrometry. The use of varying HCMV deletion mutants resulted in ahigher variability of identified HCMV-derived peptide species, anddemonstrated that HLA-I immunoevasins affect not only the quantity, butalso the quality of HLA-I antigen processing and presentation.

The present invention thus further relates to a method for identifyingHCMV-derived HLA class I ligands comprising generating a HCMV deletionmutant virus, lacking at least one functional HLA class I immunoevasin,preferably all HLA class I immunoevasins, infecting a cell culture, forexample a fibroblast cell culture, expressing at least one HLA-I type ofinterest, and isolating and identifying presented HCMV-derived HLA-Iligands from said cell culture. The ligands (peptides) can then be usedin the context of the present invention, i.e. for respective vaccines,therapies, and the generation of T-cells and/or -receptors as describedherein. Preferred is a peptide or variant according to the presentinvention, wherein said peptide consists or consists essentially of anamino acid sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 100or optionally comprises an extension of one N- and/or C-terminal,preferably naturally occurring, amino acid. More preferred is a peptideor variant according to the present invention, wherein the amino acidsequence is selected from SEQ ID NO: 1 to 4, 24 to 29, 40, 41, 51 to 55,67, 68, 80, 87 to 89, and 99 to 101.

Using this strategy, the inventors identified 368 unique HCMV-derivedHLA class I ligands representing an unexpectedly broad panel of 123 HCMVantigens. Functional characterization revealed memory T-cell responsesin seropositive individuals for a substantial proportion (28%) of thesenovel peptides. Importantly, the inventors frequently detected multipleHCMV-directed specificities in the memory T-cell pool of singleindividuals, indicating that physiological anti-HCMV T-cell responsesare directed against a broad range of antigens. Furthermore, thesememory T cells were multifunctional (IFNy, TNF) and able to exertcytolytic activity in vitro.

Thus, the unbiased identification of naturally presented viral epitopesenabled a comprehensive and systematic assessment of the physiologicalrepertoire of anti-HCMV T-cell specificities in seropositiveindividuals. This approach proved to be superior to procedures applyingin silico analysis to identify true viral antigens, and the use ofvarying HCMV deletion mutants resulted in a higher variability ofidentified HCMV-derived peptide species.

The following tables show the peptides according to the presentinvention, and their respective SEQ ID NOs.

TABLE 1 Peptide epitopes of the invention,underlying protein, sequence, actual HLA restriction (where determined).Actual HLA restriction (identified Source using protein tetramer andSequence/ staining position SEQ ID NO: and ICS) US8 74-82 GVLDAVWRVA*02:01 (SEQ ID NO: 1) UL150A 152-161 ALWDVALLEV A*02:01 (SEQ ID NO: 2)UL100 200-208 TLIVNLVEV A*02:01 (SEQ ID NO: 3) UL44 259-267 GLFA VENFLDR (SEQ ID NO: 4) UL71 40-48 FLDENFKQL DR (SEQ ID NO: 5) UL105 431-439RLFDLPVYC (SEQ ID NO: 6) UL29 175-183 RLQPNVPLV (SEQ ID NO: 7)US16 134-144 GLLAHIPALGV (SEQ ID NO: 8) US29 293-301 ALSPSTSKV(SEQ ID NO: 9) UL29 344-352 SLYEANPEL (SEQ ID NO: 10) UL86 146-154TILDKILNV (SEQ ID NO: 11) US16 186-194 TLINGVWVV (SEQ ID NO: 12)US16 134-142 GLLAHIPAL (SEQ ID NO: 13) UL48 132-141 ALYPEYIYTV(SEQ ID NO: 14) UL47 766-744 GLNERLLSV (SEQ ID NO: 15) UL34 130-138ALFNQLVFTA (SEQ ID NO: 16) UL56 124-132 FTDNVRFSV (SEQ ID NO: 17)UL128 145-153 GLDQYLESV (SEQ ID NO: 18) UL84 133-141 ALLGRLYFI(SEQ ID NO: 19) UL4 96-104 NYNEQHYRY (SEQ ID NO: 20) US27 276-284LYVGQFLAY (SEQ ID NO: 21) UL4 88-97 SFFPKLQGNY (SEQ ID NO: 22) UL4 89-97FFPKLQGNY (SEQ ID NO: 23) UL16 162-170 YPRPPGSGL B*07:02 (SEQ ID NO: 24)UL83 417-426 TPRVTGGGAM B*07:02 (SEQ ID NO: 25) TRS1 166-174 SPRDAWIVLB*07:02 (SEQ ID NO: 26) UL52 349-357 SPSRDRFVQL B*07:02 (SEQ ID NO: 27)UL23 22-30 RPWKPGQRV B*07:02 (SEQ ID NO: 28) UL46 76-84 SPRHLYISLB*07:02 (SEQ ID NO: 29) UL38 225-235 IPMTFVDRDSL (SEQ ID NO: 30)US30 313-321 RPFPSTHQL (SEQ ID NO: 31) UL83 49-57 RVSQPSLIL(SEQ ID NO: 32) UL82 245-254 SPHPPTSVFL (SEQ ID NO: 33) UL27 485-493IPDYRSVSL (SEQ ID NO: 34) UL31 310-317 APFGRVSV (SEQ ID NO: 35)TRS1/IRS1 IPVERQAL 92-99 (SEQ ID NO: 36) UL98 135-143 APNYRQVEL(SEQ ID NO: 37) UL40 210-218 LPNDHHYAL (SEQ ID NO: 38) US 12 82-89APYLRDTL (SEQ ID NO: 39) UL112/UL113 SENGNLQVTY B*44:02 125-134(SEQ ID NO: 40) UL117 358-366 HETGVYQMW B*44:02 (SEQ ID NO: 41)UL17 24-32 DEQVSKRSW B*44:02 (SEQ ID NO: 42) TRL12 402-410 SESEFIVRYB*44:02 (SEQ ID NO: 43) UL147A 51-59 EEQDYRALL (SEQ ID NO: 44)UL78 150-158 RENAGVALY (SEQ ID NO: 45) US21 71-80 AEPNFPKNVW(SEQ ID NO: 46) TRS1/IRS1 EEATALGREL 276-285 (SEQ ID NO: 47)US11 103-111 SESLVAKRY (SEQ ID NO: 48) UL54 755-763 LENGVTHRF(SEQ ID NO: 49) US22 72-81 REQAAIPQIY (SEQ ID NO: 50) UL105 715-723YADPFFLKY A*01:01 (SEQ ID NO: 51) UL44 245-253 VTEHDTLLY A*01:01(SEQ ID NO: 52) UL69 569-578 RTDPATLTAY A*01:01 (SEQ ID NO: 53)US28 122-130 ITEIALDRY A*01:01 (SEQ ID NO: 54) UL55 657-665 NTDFRVLELYA*01:01 (SEQ ID NO: 55) UL36 82-91 FVEGPGFMRY (SEQ ID NO: 56)UL148 282-290 SLDRFIVQY DR (SEQ ID NO: 57) UL25 370-379 YTSRGALYLY(SEQ ID NO: 58) UL86 1346-1354 TSETHFGNY (SEQ ID NO: 59) US34 92-101GSDALPAGLY (SEQ ID NO: 60) UL48 1607-1617 VTDYGNVAFKY (SEQ ID NO: 61)IRS1/TRS1 LLDELGAVFGY 464-474 (SEQ ID NO: 62) UL112/UL113 ISENGNLQVTY124-134 (SEQ ID NO: 63) UL105 616-624 VTDPEHLMM (SEQ ID NO: 64)UL105 360-369 DLDFGDLLKY (SEQ ID NO: 65) UL78 222-232 YSDRRDHVWSY(SEQ ID NO: 66) UL77 228-236 GLYTQPRWK A*03:01 (SEQ ID NO: 67)UL57 790-798 RVKNRPIYR A*03:01 (SEQ ID NO: 68) UL36 51-60 RSALGPFVGKA*03:01 (SEQ ID NO: 69) UL123 184-192 KLGGALQAK (SEQ ID NO: 70)US33A 13-21 KLGYRPHAK A*03:01 (SEQ ID NO: 71) US24 136-145 RVYAYDTREK(SEQ ID NO: 72) UL25 580-588 GVSSVTLLK (SEQ ID NO: 73) UL84 3-11RVDPNLRNR (SEQ ID NO: 74) UL70 698-706 SVRLPYMYK (SEQ ID NO: 75)UL79 237-245 RTFAGTLSR (SEQ ID NO: 76) UL57 1044-1052 RLADVLIKR(SEQ ID NO: 77) UL70 697-706 RSVRLPYMYK (SEQ ID NO: 78) UL122 113-121SVSSAPLNK (SEQ ID NO: 79) UL13 465-473 YLVRRPMTI B*08:01 (SEQ ID NO: 80)UL36 199-207 VMKFKETSF (SEQ ID NO: 81) UL84 239-247 TPLLKRLPL(SEQ ID NO: 82) UL40 170-178 HLKLRPATF (SEQ ID NO: 83) UL84 500-507FISSKHTL (SEQ ID NO: 84) UL44 26-34 QLRS VIRAL (SEQ ID NO: 85) UL148 1-8MLRLLFTL B*08:01 (SEQ ID NO: 86) UL83 116-123 LPLKMLNI B*51:01(SEQ ID NO: 87) UL38 156-164 FPVEVRSHV B*51:01 (SEQ ID NO: 88)UL56 503-511 DARSRIHNV B*51:01 (SEQ ID NO: 89) UL71 330-338 IPPPQIPFV(SEQ ID NO: 90) US28 158-166 IAIPHFMVV (SEQ ID NO: 91) US23 65-73IPHNWFLQV (SEQ ID NO: 92) UL33 162-170 VPAAVYTTV (SEQ ID NO: 93)UL14 66-74 FPAHDWPEV (SEQ ID NO: 94) UL122 449-457 MPVTHPPEV(SEQ ID NO: 95) UL75 540-549 FPDATVPATV (SEQ ID NO: 96) UL48 1322-1331LPYLSAERTV (SEQ ID NO: 97) UL147A2-10 SLFYRAVAL (SEQ ID NO: 98)UL26 61-69 LPYPRGYTL B*08:01/B*51:01 (SEQ ID NO: 99) B*08:01/B*51:01UL34 180-188 LPHERHREL B*08:01 (SEQ ID NO: 100)

TABLE 2 Preferred dominant epitopes of the invention,sequence, actual HLA restriction Sequence actual HLA Protein SEQ ID NO:restriction US8 74-82 GVLDAVWRV A*02:01 SEQ ID NO: 1 UL150A 152-ALWDVALLEV A*02:01 161 SEQ ID NO: 2 UL100 200-208 TLIVNLVEV A*02:01SEQ ID NO: 3 UL44 259-267 GLFAVENFL Class II SEQ ID NO: 4 UL16 162-170YPRPPGSGL* B*07:02 SEQ ID NO: 24 UL83 417-426 TPRVTGGGAM* B*07:02SEQ ID NO: 25 TRS1 166-174 SPRDAWIVL B*07:02 SEQ ID NO: 26 UL52 349-357SPSRDRFVQL B*07:02 SEQ ID NO: 27 UL23 22-30 RPWKPGQRV B*07:02SEQ ID NO: 28 UL46 76-84 SPRHLYISL B*07:02 SEQ ID NO: 29 UL112/UL113SENGNLQVTY B*44:02 125-134 SEQ ID NO: 40 UL117 358-366 HETGVYQMW B*44:02SEQ ID NO: 41 UL105 715-723 YADPFFLKY* A*01:01 SEQ ID NO: 51UL44 245-253 VTEHDTLLY* A*01:01 SEQ ID NO: 52 UL69 569-578 RTDPATLTAYA*01:01 SEQ ID NO: 53 US28 122-130 ITEIALDRY A*01:01 SEQ ID NO: 54UL55 657-665 NTDFRVLELY A*01:01 SEQ ID NO: 55 UL77 228-236 GLYTQPRWKA*03:01 SEQ ID NO: 67 UL57 790-798 RVKNRPIYR A*03:01 SEQ ID NO: 68UL34 180-188 LPHERHREL B*08:01 SEQ ID NO: 100 UL26 61-69 LPYPRGYTLB*08:01/51:01 SEQ ID NO: 99 UL13 465-473 YLVRRPMTI B*08:01 SEQ ID NO: 80UL83 116-123 LPLKMLNI* B*51:01 SEQ ID NO: 87 UL38 156-164 FPVEVRSHVB*51:01 SEQ ID NO: 88 UL26 61-69 LPYPRGYTL B*08:01/51:01 SEQ ID NO: 99UL56 503-511 DARSRIHNV B*51:01 SEQ ID NO: 89 UL83 495-503 NLVPMVATV*A*02:01 SEQ ID NO: 101

The present invention further relates to a peptide or variant thereofaccording to the present invention, wherein said peptide is modifiedand/or includes non-peptide bonds.

The present invention further relates to a peptide or variant thereofaccording to the present invention, wherein said peptide is part of afusion protein, in particular comprising the N-terminal amino acids ofthe HLA-DR antigen-associated invariant chain (li).

The present invention further relates to an antibody, in particular asoluble or membrane-bound antibody that specifically binds to thepeptide or variant thereof according to the present invention,preferably the peptide or variant thereof according to the presentinvention when bound to an MHC molecule.

The present invention further relates to a T cell receptor, preferably arecombinant, soluble or membrane-bound T cell receptor that is reactivewith an HLA ligand, wherein said ligand is at least 75% identical,preferably at least 88% identical, and most preferred 100% identical toan amino acid sequence according to the present invention.

The present invention further relates to a T cell receptor according tothe present invention, wherein said T cell receptor is provided as asoluble molecule, and optionally comprises an effector function, such asan immune stimulating domain or toxin.

The present invention further relates to a nucleic acid, encoding apeptide or variant thereof according to the present invention, theantibody according to the present invention or the T cell receptoraccording to the present invention, wherein said nucleic acid isoptionally linked to a heterologous promoter sequence.

The present invention further relates to an expression vector expressingthe nucleic acid according to the present invention.

The present invention further relates to a recombinant host cellcomprising a recombinant peptide according to the present invention, arecombinant antibody according to the present invention, a recombinant Tcell receptor according to the present invention, the nucleic acidaccording to claim 7 or the expression vector according to the presentinvention, wherein said host cell preferably is an antigen presentingcell such as a dendritic cell, or preferably is a T cell or NK cell.

The present invention further relates to a method for producing thepeptide or variant thereof according to the present invention, theantibody according to the present invention, or the T cell receptoraccording to the present invention, the method comprising culturing thehost cell according to the present invention that presents the peptideaccording to the present invention, or expresses the nucleic acidaccording to the present invention or comprises the expression vectoraccording to the present invention, and isolating said peptide orvariant thereof, said antibody or said T cell receptor from said hostcell and/or its culture medium.

The present invention further relates to an in vitro method forproducing activated T lymphocytes, the method comprising contacting invitro T cells with antigen loaded human class I or II MHC moleculesexpressed on the surface of a suitable antigen-presenting cell or anartificial construct mimicking an antigen-presenting cell for a periodof time sufficient to activate said T cells in an antigen specificmanner, wherein said antigen is a peptide according to the presentinvention.

The present invention further relates to an activated T lymphocyte,produced by the method according to the present invention thatselectively recognizes a cell which presents a polypeptide comprising anamino acid sequence as disclosed herein.

The present invention further relates to a pharmaceutical compositioncomprising at least one active ingredient selected from the groupconsisting of the peptide or variant thereof according to the presentinvention, the antibody according to the present invention, the T cellreceptor according to the present invention, the nucleic acid accordingto the present invention, the expression vector according to the presentinvention, the host cell according to the present invention or theactivated T lymphocyte according to the present invention, and apharmaceutically acceptable carrier, and optionally additionalpharmaceutically acceptable excipients and/or stabilizers.

The present invention further relates to a method for producing apersonalized anti-viral vaccine, said method comprising: a) identifyingat least one HCMV-associated peptide according to any one of SEQ ID NO:1 to SEQ ID NO: 101 in a sample from said individual patient; b)selecting at least one peptide as identified in said sample from stepa), and c) formulating the at least one peptide as selected in step b)into a personalized anti-viral vaccine.

The present invention further relates to a peptide or variant thereofaccording to the present invention, the antibody according to thepresent invention, the T cell receptor according to the presentinvention, the nucleic acid according to the present invention, theexpression vector according to the present invention, the host cellaccording to the present invention or the activated T lymphocyteaccording to the present invention, the pharmaceutical compositionaccording to the present invention, or the vaccine as produced accordingto the present invention for use in medicine.

The present invention further relates to a peptide or variant thereofaccording to the present invention, the antibody according to thepresent invention, the T cell receptor according to the presentinvention, the nucleic acid according to the present invention, theexpression vector according to the present invention, the host cellaccording to the present invention or the activated T lymphocyteaccording to the present invention, the pharmaceutical compositionaccording to the present invention, or the vaccine as produced accordingto the present invention for use in the diagnosis and/or treatment ofHCMV infection, or for use in the manufacture of a medicament againstHCMV infection.

The present invention further relates to a peptide or variant thereofaccording to the present invention, the antibody according to thepresent invention, the T cell receptor according to the presentinvention, the nucleic acid according to the present invention, theexpression vector according to the present invention, the host cellaccording to the present invention or the activated T lymphocyteaccording to the present invention, the pharmaceutical compositionaccording to the present invention, or the vaccine as produced accordingto the present invention for use according to the present invention,wherein said HCMV infection exhibits a co-morbidity with cancer,inflammatory diseases, hypertensive diseases, and pulmonary diseases.

The present invention further relates to a kit comprising: a) acontainer comprising a peptide or variant thereof according to thepresent invention, the antibody according to the present invention, theT cell receptor according to the present invention, the nucleic acidaccording to the present invention, the expression vector according tothe present invention, the host cell according to the present invention,or the activated T lymphocyte according to the present invention, thepharmaceutical composition according to the present invention, or thevaccine as produced according to the present invention, in solution orin lyophilized form; b) optionally, a second container containing adiluent or reconstituting solution for the lyophilized formulation; c)optionally, at least one additional peptide selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 101, and d) optionally,instructions for (i) use of the solution or (ii) reconstitution and/oruse of the lyophilized formulation, and e) a substance or combination ofsubstances acting as an adjuvant, i.e. acting as an inducer of immuneresponses Preferred is the kit according to the present invention,further comprising one or more of (iii) a buffer, (iv) a diluent, (v) afilter, (vi) a needle, or (v) a syringe, or vi) a mixing device.

Finally, the present invention further relates to a method for treatingHCMV infection in target cells in a patient, wherein said target cellspresent at least one peptide comprising an amino acid sequence accordingto the present invention, comprising administering to said patient aneffective amount of activated T lymphocytes according to the presentinvention, the pharmaceutical composition according to the presentinvention, and/or of the vaccine as produced according to the presentinvention.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in literature (Brossart and Bevan, 1997; Rock et al.,1990). MHC class II molecules can be found predominantly on professionalantigen presenting cells (APCs), and primarily present peptides ofexogenous or transmembrane proteins that are taken up by APCs e.g.during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR.

It is well known that the TCR, the peptide and the MHC are therebypresent in a stoichiometric amount of 1:1:1.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-l- binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove. In the MHC class I dependent immunereaction, peptides not only have to be able to bind to certain MHC classI molecules expressed by virally infected cells, they subsequently alsohave to be recognized by T cells bearing specific T cell receptors(TCR).

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of virus associated antigens has raised the possibilityof using a host's immune system to intervene with viral infection.Various mechanisms of harnessing both the humoral and cellular arms ofthe immune system are currently being explored for immunotherapy.Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying infected cells. CD8-positiveT-cells in particular, which recognize class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 10amino acid residues derived from proteins or defect ribosomal products(DRIPS) located in the cytosol, play an important role in the response.The MHC-molecules of the human are also designated as humanleukocyte-antigens (HLA).

The term “T-cell response” shall relate to the specific proliferationand activation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, or 13 amino acids orlonger, and in case of MHC class II peptides (longer variants of thepeptides of the invention) they can be as long as 14, 15, 16, 17, 18, 19or 20 or more amino acids in length. The peptides can be extended by oneamino acid on their N-and/or C-terminus, these extensions of courseshould not substantially interfere with the activity of those peptides.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

Consequently, as used herein, “a pharmaceutically acceptable salt”refers to a derivative of the disclosed peptides wherein the peptide ismodified by making acid or base salts of the agent. For example, acidsalts are prepared from the free base (typically wherein the neutralform of the drug has a neutral —NH2 group) involving reaction with asuitable acid. Suitable acids for preparing acid salts include bothorganic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methane sulfonic acid, ethane sulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like, as well asinorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid phosphoric acid and the like. Conversely, preparationof basic salts of acid moieties which may be present on a peptide areprepared using a pharmaceutically acceptable base such as sodiumhydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide,trimethylamine or the like. In an especially preferred embodiment, thepeptides are—and the pharmaceutical compositions comprise the peptidesas—salts of acetic acid (acetates), trifluoro acetates or hydrochloricacid (chlorides).

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 101and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl—, Br—, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻, and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺,Ca²⁺, Mn²⁺, Cu²⁺, and Ba²⁺. Particularly salts are selected from(NH₄)₃PO₄, (NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br,NH₄NO₃, NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄,Rb₄CH₃COO, Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄,KH₂PO₄, K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄,Na₂HPO₄, NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, Nal,NaSCN, ZnCI₂ Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCI, CsBr,CsNO₃, CsClO₄, CSI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO,LiCl, LiBr, LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄,Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂,MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄)₂, Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄,Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂,Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄, Ba(CH₃COO)₂, BaCI₂, BaBr₂,Ba(NO₃)₂, Ba(CIO₄)₂, BaI₂, and Ba(SCN)₂. Particularly preferred are NHacetate, MgCl₂, KH₂PO, Na₂SO, KCl, NaCl, and CaCl₂, such as, forexample, the chloride or acetate (trifluoroacetate) salts.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides. The nucleotide sequence codingfor a particular peptide, oligopeptide, or polypeptide may be naturallyoccurring or they may be synthetically constructed. Generally, DNAsegments encoding the peptides, polypeptides, and proteins of thisinvention are assembled from cDNA fragments and short oligonucleotidelinkers, or from a series of oligonucleotides, to provide a syntheticgene that is capable of being expressed in a recombinant transcriptionalunit comprising regulatory elements derived from a microbial or viraloperon. As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene. The coding region can be derivedfrom a non-mutated (“normal”), mutated or altered gene, or can even bederived from a DNA sequence, or gene, wholly synthesized in thelaboratory using methods well known to those of skill in the art of DNAsynthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a sequence as claimed or described after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula: percentidentity=100[1−(C/R)] wherein C is the number of differences between theReference Sequence and the Compared Sequence over the length ofalignment between the Reference Sequence and the Compared Sequence,wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and (iv) the alignment has to start at position1 of the aligned sequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity. A sequence identity can be determined by creating an alignmentusing, for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 101. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells. These T cells can subsequently cross-react with cellsand kill cells that express a polypeptide that contains the naturalamino acid sequence of the cognate peptide as defined in the aspects ofthe invention. As can be derived from the scientific literature anddatabases (Rammensee et al., 1999; Godkin et al., 1997), certainpositions of HLA binding peptides are typically anchor residues forminga core sequence fitting to the binding motif of the HLA receptor, whichis defined by polar, electrophysical, hydrophobic and spatial propertiesof the polypeptide chains constituting the binding groove. Thus, oneskilled in the art would be able to modify the amino acid sequences setforth in SEQ ID NO: 1 to SEQ ID NO: 101, by maintaining the known anchorresidues, and would be able to determine whether such variants maintainthe ability to bind MHC class I or II molecules. The variants of thepresent invention retain the ability to bind to the TCR of activated Tcells, which can subsequently cross-react with and kill cells thatexpress a polypeptide containing the natural amino acid sequence of thecognate peptide as defined in the aspects of the invention. The original(unmodified) peptides as disclosed herein can be modified by thesubstitution of one or more residues at different, possibly selective,sites within the peptide chain, if not otherwise stated. Preferablythose substitutions are located at the end of the amino acid chain. Suchsubstitutions may be of a conservative nature, for example, where oneamino acid is replaced by an amino acid of similar structure andcharacteristics, such as where a hydrophobic amino acid is replaced byanother hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.” Variants are further length variants, where aminoacid(s) are added to the “core sequence” of the amino acid sequences setforth in SEQ ID NO: 1 to SEQ ID NO: 101.

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1 -small aliphatic, nonpolar orslightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar,negatively charged residues and their amides (Asp, Asn, Glu, Gin); Group3-polar, positively charged residues (His, Arg, Lys); Group 4-large,aliphatic, nonpolar residues (Met, Leu, lie, Val, Cys); and Group5-large, aromatic residues (Phe, Tyr, Trp). If substitutions at morethan one position are found to result in a peptide with substantiallyequivalent or greater antigenic activity as defined herein, thencombinations of those substitutions will be tested to determine if thecombined substitutions result in additive or synergistic effects on theantigenicity of the peptide. At most, no more than four positions withinthe peptide would be simultaneously substituted. The amino acid residuesthat do not substantially contribute to interactions with the T-cellreceptor can be modified by replacement with other amino acid whoseincorporation does not substantially affect T-cell reactivity and doesnot eliminate binding to the relevant MHC.

It is possible that MHC class I epitopes, although usually between 8 and12 amino acids long, are generated by peptide processing from longerpeptides or proteins that include the actual epitope. It is preferredthat the residues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing. The peptides of the invention can beelongated by up to four amino acids, that is 1 , 2, 3 or 4 amino acidscan be added to either end in any combination between 4:0 and 0:4. Theamino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides. Thus, the epitopes of the present invention may be identicalto naturally occurring tumor-associated or tumor-specific epitopes ormay include epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity. Of course, the peptide or variant according to thepresent invention will have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class I or II. Binding of apeptide or a variant to a MHC complex may be tested by methods known inthe art.

A particularly preferred embodiment of the invention relates to thepeptide or variant according to the present invention, wherein saidpeptide consists or consists essentially of an amino acid sequenceaccording to any of SEQ ID NO: 1 to SEQ ID NO: 100 or optionallycomprises an extension of one N- and/or C-terminal amino acid.

A particularly preferred embodiment of the invention relates to thepeptide or variant according to the present invention, wherein saidpeptide comprises an immune dominant epitope, wherein the amino acidsequence is selected from SEQ ID NO: 1 to 4, 24 to 29, 40, 41, 51 to 55,67, 68, 80, 87 to 89, and 99 to 101. In another particularly preferredembodiment of the invention the peptide then consists or consistsessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 101. “Consisting essentially of” shall mean that a peptideaccording to the present invention, in addition to the sequenceaccording to any of SEQ ID NO: 1 to SEQ ID NO 101 or a variant thereofcontains additional N- and/or C-terminally located stretches of aminoacids that are not necessarily forming part of the peptide thatfunctions as an epitope for MHC molecules.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “li”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds. In a reverse peptide bond,the amino acid residues are not joined by peptide (—CO—NH—) linkages butthe peptide bond is reversed. Such retro-inverso peptidomimetics may bemade using methods known in the art, for example such as those describedin Meziere et al (1997) (Meziere et al., 1997). This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al. (Meziere et al., 1997)show that for MHC binding and T helper cell responses, thesepseudopeptides are useful. Retro-inverse peptides, which contain NH—CObonds instead of CO—NH peptide bonds, are much more resistant toproteolysis. A non-peptide bond is, for example, —CH₂—NH, —CH₂S—,—CH₂CH₂—, —CH═CH—, —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No.4,897,445 provides a method for the solid phase synthesis of non-peptidebonds (—CH₂—NH) in polypeptide chains which involves polypeptidessynthesized by standard procedures and the non-peptide bond synthesizedby reacting an amino aldehyde and an amino acid in the presence ofNaCNBH₃.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981).

Another aspect of the present invention relates to an antibody, inparticular a soluble or membrane-bound antibody that specifically bindsto the peptide or variant thereof according to the present invention,preferably the peptide or variant thereof according to the presentinvention when bound to an MHC I or II molecule.

A further aspect of the invention provides an antibody that specificallybinds to a human major histocompatibility complex (MHC) class I or IIbeing complexed with a HLA-restricted antigen, wherein the antibodypreferably is a polyclonal antibody, monoclonal antibody, bi-specificantibody and/or a chimeric antibody.

It is a further aspect of the invention to provide a method forproducing a recombinant antibody specifically binding to a human majorhistocompatibility complex (MHC) class I or II being complexed with aHLA-restricted antigen, the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically binding to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen. Other methods for producing such antibodies andsingle chain class I major histocompatibility complexes, as well asother tools for the production of these antibodies are disclosed, forexample, in WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, andin publications (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg etal., 2003), which for the purposes of the present invention are allexplicitly incorporated by reference in their entireties. Preferably,the antibody is binding with a binding affinity of below 20 nanomolar,preferably of below 10 nanomolar, to the complex, which is also regardedas “specific” in the context of the present invention.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention. Furthermore, the peptides whencomplexed with the respective MHC can be used for the production ofantibodies and/or TCRs, in particular sTCRs, according to the presentinvention, as well. Respective methods are well known to the person ofskill, and can be found in the respective literature as well.

It is a further aspect of the invention to provide a T cell receptor,preferably a recombinant, soluble or membrane-bound T cell receptor,that is reactive with an HLA ligand, such as, for example, a peptideaccording to the present invention, wherein said ligand is at least 75%identical, preferably at least 88% identical, and most preferred 100%identical to an amino acid sequence according to the present invention.Preferably, the T cell receptor according to the present invention isprovided as a soluble molecule, and optionally comprises an effectorfunction, such as an immune stimulating domain or toxin.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane. With respectto gamma/delta TCRs, the term “TCR gamma variable domain” as used hereinrefers to the concatenation of the TCR gamma V (TRGV) region withoutleader region (L), and the TCR gamma J (TRGJ) region, and the term TCRgamma constant domain refers to the extracellular TRGC region, or to aC-terminal truncated TRGC sequence. Likewise, the term “TCR deltavariable domain” refers to the concatenation of the TCR delta V (TRDV)region without leader region (L) and the TCR delta D/J (TRDD/TRDJ)region, and the term “TCR delta constant domain” refers to theextracellular TRDC region, or to a C-terminal truncated TRDC sequence.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a viral infection based on a biopsied or other suitablesample.

The antibodies or TCRs may also be used for in vitro or in vivodiagnostic assays. Generally, the antibody is labeled with aradionucleotide (such as ¹¹¹ln, ⁹⁹Tc, ¹⁴C, ¹³¹l ³H, ³²P or ³⁵S) so thatthe tumor can be localized using immunoscintiography. In one embodiment,antibodies or fragments thereof bind to the extracellular domains of twoor more targets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM. Antibodies for diagnostic use may be labeled with probes suitablefor detection by various imaging methods. Methods for detection ofprobes include, but are not limited to, fluorescence, light, confocaland electron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis. The present description furtherrelates to a method of identifying and isolating a TCR according to thepresent description, said method comprising obtaining a transgenic mousewith the entire human TCRab gene loci (1.1 and 0.7 Mb), whose T-cellsexpress a diverse human TCR repertoire that compensates for mouse TCRdeficiency, immunizing the mouse with peptide, incubating PBMCs obtainedfrom the transgenic mice with tetramer-phycoerythrin (PE), and isolatingthe high avidity T-cells by fluorescence activated cell sorting(FACS)-Calibur analysis.

In one embodiment, the present invention provides a method of producinga TCR as described herein, the method comprising culturing a host cellcapable of expressing the TCR under conditions suitable to promoteexpression of the TCR. In one additional aspect, to obtain T-cellsexpressing TCRs of the present description, nucleic acids encodingTCR-alpha and/or TCR-beta chains of the present description are clonedinto expression vectors, such as gamma retrovirus or lentivirus. Therecombinant viruses are generated and then tested for functionality,such as antigen specificity and functional avidity. An aliquot of thefinal product is then used to transduce the target T-cell population(generally purified from patient-PBMCs), which is expanded beforeinfusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentinvention, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains. The alpha and beta chains of a TCR of the presentinvention may be encoded by nucleic acids located in separate vectors,or may be encoded by polynucleotides located in the same vector.Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of overcoming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

The present invention further relates to a nucleic acid, encoding apeptide or variant thereof according to the present invention, theantibody according to the present invention or the T cell receptoraccording to the present invention, wherein said nucleic acid isoptionally linked to a heterologous promoter sequence. The presentinvention further relates to the nucleic acid according to the presentinvention that is DNA, cDNA, PNA, RNA or combinations thereof. Thepresent invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention. In a preferred embodiment, the term “nucleotide sequence”refers to a heteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides and othermolecules of the invention, such as TCRs and antibodies, polypeptides,and proteins of this invention are assembled from cDNA fragments andshort oligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic gene that is capable of being expressed in arecombinant transcriptional unit comprising regulatory elements derivedfrom a microbial or viral operon.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence. The term “coding region” refers to that portion of a genewhich either naturally or normally codes for the expression product ofthat gene in its natural genomic environment, i.e., the region coding invivo for the native expression product of the gene.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the molecule of theinvention including artificial (man-made) start and stop codonscompatible for the biological system the sequence is to be expressed by,for example, a dendritic cell or another cell system useful for theproduction of TCRs. The coding region can be derived from a non-mutated(“normal”), mutated or altered gene, or can even be derived from a DNAsequence, or gene, wholly synthesized in the laboratory using methodswell known to those of skill in the art of DNA synthesis. The term“expression product” means the polypeptide or protein that is thenatural translation product of the gene and any nucleic acid sequencecoding equivalents resulting from genetic code degeneracy and thuscoding for the same amino acid(s). The term “fragment”, when referringto a coding sequence, means a portion of DNA comprising less than thecomplete coding region, whose expression product retains essentially thesame biological function or activity as the expression product of thecomplete coding region. The term “promoter” means a region of DNAinvolved in binding of RNA polymerase to initiate transcription.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules. Synthetic linkerscontaining one or more restriction sites provide an alternative methodof joining the DNA segment to vectors. Synthetic linkers containing avariety of restriction endonuclease sites are commercially availablefrom a number of sources including International Biotechnologies Inc.New Haven, Conn., USA. A desirable method of modifying the DNA encodingthe polypeptide of the invention employs the polymerase chain reactionas disclosed by Saiki R K, et al. (Saiki et al., 1988). This method maybe used for introducing the DNA into a suitable vector, for example byengineering in suitable restriction sites, or it may be used to modifythe DNA in other useful ways as is known in the art. If viral vectorsare used, pox- or adenovirus vectors are preferred.

Nucleic acids encoding molecules, such as TCRs or antibodies, of thepresent description may be codon optimized to increase expression from ahost cell. Redundancy in the genetic code allows some amino acids to beencoded by more than one codon, but certain codons are less “optimal”than others because of the relative availability of matching tRNAs aswell as other factors (Gustafsson et al., 2004). Modifying the, forexample, TCR-alpha and TCR-beta gene sequences such that each amino acidis encoded by the optimal codon for mammalian gene expression, as wellas eliminating mRNA instability motifs or cryptic splice sites, has beenshown to significantly enhance TCR-alpha and TCR-beta gene expression(Scholten et al., 2006).

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance. Alternatively, the gene for such selectable traitcan be on another vector, which is used to co-transform the desired hostcell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the molecule of theinvention, which can then be recovered. Many expression systems areknown, including bacteria (for example E. coli and Bacillus subtilis),yeasts (for example Saccharomyces cerevisiae), filamentous fungi (forexample Aspergillus spec), plant cells, animal cells and insect cells.Preferably, the system can be mammalian cells such as CHO cellsavailable from the ATCC Cell Biology Collection. A typical mammaliancell vector plasmid for constitutive expression comprises the CMV orSV40 promoter with a suitable poly A tail and a resistance marker, suchas neomycin.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, or may be linked without any additional peptide(s) between them.These constructs can also be used for antiviral therapy, and may induceimmune responses both involving MHC I and MHC II.

The present invention also relates to a host cell transformed ortransfected with a polynucleotide vector construct of the presentinvention. The host cell can be either prokaryotic or eukaryotic.Bacterial cells may be preferred prokaryotic host cells in somecircumstances and typically are a strain of E. coli such as, forexample, the £. coli strains DH5. Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501.Preferred mammalian host cells include Chinese hamster ovary (CHO)cells, NIH Swiss mouse embryo cells NIH/3T3, monkey kidney-derived COS-1cells, and 293 cells which are human embryonic kidney cells. Preferredinsect cells are Sf9 cells which can be transfected with baculovirusexpression vectors. An overview regarding the choice of suitable hostcells for expression can be found in literature known to the person ofskill. Transformation of appropriate cell hosts with a DNA construct ofthe present invention is accomplished by well-known methods thattypically depend on the type of vector used. With regard totransformation of prokaryotic host cells, see, for example, Cohen et al.(Cohen et al., 1972) and (Green and Sambrook, 2012).

Transformation of yeast cells is described in Sherman et al. (Sherman etal., 1986). With regard to vertebrate cells, reagents useful intransfecting such cells, for example calcium phosphate and DEAE-dextranor liposome formulations, are available from Stratagene Cloning Systems,or Life Technologies Inc., Gaithersburg, Md. 20877, USA. Electroporationis also useful for transforming and/or transfecting cells and is wellknown in the art for transforming yeast cell, bacterial cells, insectcells and vertebrate cells.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a molecule of the invention,e.g. comprising the peptide or variant of the invention. Thus, the DNA,preferably encoding the peptide or variant of the invention, may be usedin accordance with known techniques, appropriately modified in view ofthe teachings contained herein, to construct an expression vector, whichis then used to transform an appropriate host cell for the expressionand production of the polypeptide of the invention. The DNA (or in thecase of retroviral vectors, RNA) encoding a polypeptide constituting themolecule of the invention may be joined to a wide variety of other DNAsequences for introduction into an appropriate host. The companion DNAwill depend upon the nature of the host, the manner of the introductionof the DNA into the host, and whether episomal maintenance orintegration is desired.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies. It will be appreciatedthat certain host cells of the invention are useful in the preparationof the molecules of the invention, for example bacterial, yeast andinsect cells. However, other host cells may be useful in certaintherapeutic methods. For example, antigen-presenting cells, such asdendritic cells, may usefully be used to express the peptides of theinvention such that they may be loaded into appropriate MHC molecules.Thus, the current invention provides a host cell comprising a nucleicacid or an expression vector according to the invention. In a preferredembodiment, the host cell is an antigen presenting cell, in particular adendritic cell or antigen presenting cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide or other molecule of the invention from said hostcell or its culture medium. The present invention further relates tosaid method according to the present invention, wherein the peptideantigen is loaded onto class I or II MHC molecules expressed on thesurface of a suitable antigen-presenting cell or artificialantigen-presenting cell by contacting a sufficient amount of the antigenwith an antigen-presenting cell. The present invention further relatesto the method according to the present invention, wherein saidantigen-presenting cell comprises an expression vector capable ofexpressing or expressing said peptide containing SEQ ID NO: 1 to SEQ IDNO: 101. Another aspect of the present invention includes an in vitromethod for producing activated T cells, the method comprising contactingin vitro T cells with antigen loaded human MHC molecules expressed onthe surface of a suitable antigen-presenting cell for a period of timesufficient to activate the T cell in an antigen specific manner, whereinthe antigen is a peptide according to the invention. Preferably asufficient amount of the antigen is used with an antigen-presentingcell.

Another aspect of the invention relates to an activated T lymphocyte,produced by the method according to the present invention thatselectively recognizes a cell which presents a polypeptide comprising anepitope amino acid sequence as disclosed herein. The activated T cellsthat are directed against the peptides of the invention are useful intherapy. Thus, a further aspect of the invention provides activated Tcells obtainable by the foregoing methods of the invention. Activated Tcells, which are produced by the above method, will selectivelyrecognize a cell that aberrantly expresses a polypeptide that comprisesan amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 101. Preferably,the T cell recognizes the cell by interacting through its TCR with theHLA/peptide-complex (for example, binding). The T cells of the presentinvention may be used as active ingredients of a therapeuticcomposition.

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofviral infection. Any molecule of the invention, i.e. the peptide,nucleic acid, antibody, expression vector, cell, activated T cell,T-cell receptor or the nucleic acid encoding these, is useful for thetreatment of disorders, characterized by cells escaping an immuneresponse. Therefore, any molecule of the present invention may be usedas medicament or in the manufacture of a medicament. The molecule may beused by itself or combined with other molecule(s) of the invention or(a) known molecule(s).

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising at least one active ingredient selected from thegroup consisting of the peptide or variant thereof according to thepresent invention, the antibody according to the present invention, theT cell receptor according to the present invention, the nucleic acidaccording to the present invention, the expression vector according tothe present invention, the host cell according to the present inventionor the activated T lymphocyte according to the present invention, and apharmaceutically acceptable carrier, and optionally additionalpharmaceutically acceptable excipients and/or stabilizers. A“pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines. The pharmaceutical composition may be prepared forintravenous (i.v.) injection, sub-cutaneous (s.c.) injection,intradermal (i.d.) injection, intraperitoneal (i.p.) injection,intramuscular (i.m.) injection.

More preferably, the pharmaceutical composition is in the form of apeptide vaccine. Methods for formulating peptide vaccines are known tothe person of skill and disclosed in the respective literature. It maybe administered directly into the patient, into the affected organ orsystemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo tocells derived from the patient or a human cell line which aresubsequently administered to the patient, or used in vitro to select asubpopulation of immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2. Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145). The peptide may also be tagged, maybe a fusion protein, or may be a hybrid molecule. The peptides whosesequence is given in the present invention are expected to stimulate CD4or CD8 T cells. However, stimulation of CD8 T cells is more efficient inthe presence of help provided by CD4 T-helper cells. Thus, for MHC ClassI epitopes that stimulate CD8 T cells the fusion partner or sections ofa hybrid molecule suitably provide epitopes which stimulate CD4-positiveT cells. CD4- and CD8-stimulating epitopes are well known in the art andinclude those identified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID NO: 1 to SEQ ID NO: 101, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I or II molecules.

The vaccine of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLRS ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon- alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants, such as Freund's or GM-CSF, are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Several cytokines have been directly linked toinfluencing dendritic cell migration to lymphoid tissues (e.g., TNF-),accelerating the maturation of dendritic cells into efficientantigen-presenting cells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4)and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23, IL-7,IFN-alpha. IFN-beta) (Gabrilovich et al., 1996). CpG immunostimulatoryoligonucleotides have also been reported to enhance the effects ofadjuvants in a vaccine setting.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). In an aspect, a peptide described herein is in the form of apharmaceutically acceptable salt. In another aspect, a peptide in theform of a pharmaceutical salt is in crystalline form. A pharmaceuticallyacceptable salt described herein refers to salts which possess toxicityprofiles within a range that is acceptable for pharmaceuticalapplications.

The present invention further relates to a method for producing apersonalized anti-viral vaccine, said method comprising: a) identifyingat least one HCMV-associated peptide according to any one of SEQ ID NO:1 to SEQ ID NO: 101 in a sample from said individual patient; b)selecting at least one peptide as identified in said sample from stepa), and c) formulating the at least one peptide as selected in step b)into a personalized anti-viral vaccine.

In said method, for an individual patient at least one peptide selectedfrom a selection of pre-screened epitope peptides is selected forsuitability in the individual patient. The method could also be adaptedto produce T cell clones for down-stream applications, such as TCRisolations, or soluble antibodies, and other treatment options. In anaspect, the peptides are pre-screened for immunogenicity before beingincluded in the warehouse. By way of example, and not limitation, theimmunogenicity of the peptides included in the selection is determinedby a method comprising in vitro T-cell priming through repeatedstimulations of CD8+ T cells from healthy donors with artificial antigenpresenting cells loaded with peptide/MHC complexes and anti-CD28antibody.

In contrast to multi-peptide cocktails with a fixed composition ascurrently developed, the selection allows a significantly highermatching of the actual presentation of antigens with the vaccine.Selected single or combinations of several “off-the-shelf peptides willbe used for each patient in a multitarget approach. In an aspect, thepeptides are selected for inclusion in the vaccine based on theirsuitability for the individual patient based on the method according tothe present invention as described herein, or as below. The HLAphenotype, transcriptomic and peptidomic data is gathered from thepatient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing selection andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patient and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs. In addition to, or as an alternative to,selecting peptides using a selection (database) model, peptides may beidentified in the patient de novo, and then included in the vaccine.Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO. Each peptide to beincluded into a product is dissolved in DMSO. The concentration of thesingle peptide solutions has to be chosen depending on the number ofpeptides to be included into the product. For example, the singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of about 2.5 mg/ml per peptide.

The present invention further relates to the peptide or variant thereofaccording to the invention, the antibody according to the invention, theT cell receptor according to the invention, the nucleic acid accordingto the invention, the expression vector according to the invention, thehost cell according to the invention or the activated T lymphocyteaccording to the invention, the pharmaceutical composition according tothe invention, or the vaccine as produced according to the invention foruse in medicine.

The peptides of the present invention as well as other molecules of theinvention (such as cells, antibodies and TCRs) are useful for generatingan immune response in a patient by which virally infected cells can bedestroyed. An immune response in a patient can be induced by directadministration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against infected cells because the target peptides ofthe present invention are not presented on normal tissues in comparablecopy numbers, preventing the risk of undesired autoimmune reactionsagainst normal cells in the patient.

The present invention further relates to the peptide or variant thereofaccording to the invention, the antibody according to the invention, theT cell receptor according to the invention, the nucleic acid accordingto the invention, the expression vector according to the invention, thehost cell according to the invention or the activated T lymphocyteaccording to the invention, the pharmaceutical composition according tothe invention, or the vaccine as produced according to the invention foruse in the diagnosis (e.g. as above) and/or treatment of HCMV infection,or for use in the manufacture of a medicament against HCMV infection.

In addition to being useful for treating infection, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from infected cells and since it was determined thatthese peptides are not or at lower levels present in normal tissues,these peptides can be used to diagnose the presence of a viralinfection. The presence of claimed peptides on tissue biopsies in bloodsamples can assist a pathologist in diagnosis of viral infection.Detection of certain peptides by means of antibodies, mass spectrometryor other methods known in the art can tell the pathologist that thetissue sample is infected, or can be used as a biomarker for HCMV.Presence of groups of peptides can enable classification orsub-classification of diseased tissues.

The present invention further relates to the peptide or variant thereofaccording to the invention, the antibody according to the invention, theT cell receptor according to the invention, the nucleic acid accordingto the invention, the expression vector according to the invention, thehost cell according to the invention or the activated T lymphocyteaccording to the invention, the pharmaceutical composition according tothe invention, or the vaccine as produced according to the invention foruse according to the invention, wherein said HCMV infection exhibits aco-morbidity with cancer, inflammatory diseases, hypertensive diseases,and pulmonary diseases. This aspect involves co-treatment of theinfection with other suitable pharmaceuticals that are known to theperson of skill.

Another aspect then relates to a method for treating HCMV infection intarget cells in a patient, wherein said target cells present at leastone peptide comprising an amino acid sequence according to theinvention, comprising administering to said patient an effective amountof activated T lymphocytes according to the invention, thepharmaceutical composition according to the invention, and/or of thevaccine as produced according to the invention.

The molecules of the invention, like the antibodies, TCRs, nucleicacids, peptides or cells can be administered to the subject, patient, orcell by injection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The molecules of theinvention, like the antibodies, TCRs, nucleic acids, peptides or cellsmay also be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the molecules may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 {circumflex over ( )} g/kg to up to 100 mg/kgof body weight or more per day, depending on the factors mentionedabove. Following administration of an antibody, preferably for treatingviral infection, the efficacy of the therapeutic antibody can beassessed in various ways well known to the skilled practitioner. Atherapeutically-administered molecule, such as a peptide or antibody,that arrests viral infection, and/or prevents the development of newlyinfected cells, compared to the disease course that would occur in theabsence of molecule (e.g. peptide or antibody) administration, is anefficacious molecule (e.g. peptide or antibody) for treatment of viralinfection.

Another aspect then relates to a therapeutic or diagnostic kitcomprising: a) a container comprising a peptide or variant thereofaccording to the invention, the antibody according to the invention, theT cell receptor according to the invention, the nucleic acid accordingto the invention, the expression vector according to the invention, thehost cell according to the invention, or the activated T lymphocyteaccording to the invention, the pharmaceutical composition according tothe invention, or the vaccine as produced according to the invention, insolution or in lyophilized form; b) optionally, a second containercontaining a diluent or reconstituting solution for the lyophilizedformulation; c) optionally, at least one additional peptide selectedfrom the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and d)optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation. The kit mayfurther comprise one or more of (iii) a buffer, (iv) a diluent, (v) afilter, (vi) a needle, or (v) a syringe. The container is preferably abottle, a vial, a syringe or test tube; and it may be a multi-usecontainer. The pharmaceutical composition is preferably lyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials, such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration. The container holding the formulationmay be a multi-use vial, which allows for repeat administrations (e.g.,from 2-6 administrations) of the reconstituted formulation. The kit mayfurther comprise a second container comprising a suitable diluent (e.g.,sodium bicarbonate solution). Upon mixing of the diluent and thelyophilized formulation, the final peptide concentration in thereconstituted formulation is preferably at least 0.15 mg/mL/peptide (=75μg) and preferably not more than 3 mg/mL/peptide (=1500 μg). The kit mayfurther include other suitable materials, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may comprise a single container thatcontains the formulation of the pharmaceutical composition(s) accordingto the present invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay comprise distinct containers for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container. The container of a therapeutic kit may bea vial, test tube, flask, bottle, syringe, or any other means ofenclosing a solid or liquid. Usually, when there is more than onecomponent, the kit will contain a second vial or other container, whichallows for separate dosing. The kit may also contain another containerfor a pharmaceutically acceptable liquid. Preferably, a therapeutic kitwill contain an apparatus (e.g., one or more needles, syringes, eyedroppers, pipette, etc.), which enables administration of the agents ofthe invention that are components of the present kit. In addition totherapeutic kits, diagnostic kits may contain labelled compounds of theinvention, as well as materials suitable for detection in the context ofa diagnostic method.

The present formulation can be one that is suitable for administrationof the peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c, and most preferablyi.d. administration may be by infusion pump.

Various immune evasion strategies by HCMV strongly interfere with theHLA-I presentation of viral peptides on the host cell surface. However,about 10% of the memory T-cell compartment of seropositive individualsconsists of HCMV-specific T cells (18) and this number can dramaticallyrise in elderly (38).

With the inventors' approach the inventors were able to bypass immuneevasion by the US6 family genes and, for the first time, show the directisolation and identification of physiologically relevant HCMV-specificHLA ligands by mass spectrometry. The inventors' data clearly confirmthat memory T cell responses towards peptides derived from viralproteins of all expression stages and functional areas can be detectedin immunocompetent HCMV carriers. The fact that memory T cells specificfor a significant proportion of these ligands are present inHCMV-seropositive individuals clearly indicates priming of naïve T cellsagainst these antigens at any time during infection.

By now, the initial perception that HCMV T-cell responses are directedonly against few immunodominant antigens has been challenged by severalgroups. Using predictive bioinformatics and functional T cell assays,Elkington et al. identified numerous novel HCMV peptide epitopes from aheterogeneous group of 14 preselected proteins (pp28, pp50, pp65, pp150,pp71, gH, gB, IE1, US2, US3, US6, US11, UL16, and UL18) as T celltargets (16). Sylwester et al. analyzed overlapping 15-mer peptides from213 HCMV open reading frames (ORFs) by cytokine flow cytometry. Of thetested ORFs, 70% were shown to be immunogenic for CD4 and/or CD8 Tcells. Immunogenicity was influenced only modestly by ORF expressionkinetics and function (18). Interestingly, besides pp65 and IE1, UL48was the only tested ORF recognized by CD8+ T cells of more than 50% oftested donors. In contrast to that particular finding, from a total of103 identified immunogenic HCMV-derived peptides, 26 epitopes,representing 23 different proteins, were classified as dominant in theinventors' study. The inventors found immunogenic peptides from 65 HCMVproteins, confirming previous findings that HCMV-specific CD8+ T-cellimmunity in healthy virus carriers is based on a broad repertoire ofHCMV antigens. The inventors were able to validate seven previouslyknown dominant epitopes, but some well-established epitopes such asVLEETSVML_(UL123) (A*02) (SEQ ID NO: 112), YSEHPTFTSQY_(UL83) (A*01)(SEQ ID NO: 109), RPHERNGFTVL_(UL83) (B*07) (SEQ ID NO: 102), andELRRKMMYM_(UL123) (B*08) (SEQ ID NO: 103) were not detected in theinventors' assays. While the inventors' approach has technical limitsalso other reasons for the lack of detection of some dominant epitopesare imaginable. The HLA-I antigen presentation pathway could beinsufficient for processing of specific peptides in the infectedfibroblasts. To assess this, in future studies the inventors will applyIFNγ to induce the HLA-I antigen presentation machinery prior toinfection, as IFNγ induction strongly improves antigen presentation toCD8+ T cells (39). In addition, ligandome analysis at differenttime-points during infection could yield peptides of different quality.The inventors observed strongly induced numbers of HLA-B*44:02 ligandsin ΔUS2-6 infected cells, i.e. in the presence of US11 expression. Thiswas indeed surprising and the inventors have begun to address themolecular mechanisms behind this phenomenon. Thus, immunoevasins do notonly affect the efficiency, but also the quality of antigenpresentation. Therefore, it should be taken into account, thatexpression of one or combinations of several immunoevasins could resultin different HLA-I ligandome qualities. Finally, it cannot be excludedthat the ΔUS2-11 deletion mutant virus still express factors thatinterfere with HLA-I peptide loading and presentation.

The broad CD8+ T-cell response against HCMV detected in healthy donorsclearly shows that inhibition of HLA-I antigen presentation byimmunoevasins is not sufficient to prevent the induction of CD8+ T cells(17), emphasizing the role of priming of CD8+ T cells throughcross-presentation (40). However, HCMV will have a major impact, notonly quantitatively, but also qualitatively on antigen processing andpresentation in the infected cells. This could partly explain why alarge portion of HCMV-derived ligands fail to elicit a memory responsein seropositive donors. Also, different expression/presentation patternsin different cell types could have an effect on memory responses (41,42). Such non-immunogenic ligands might not be processed and presentedduring an infection in vivo, or are not recognized by naïve T cells.Furthermore, donors might lack specific naïve T cells leaving a hole inthe T-cell repertoire (43-45). Despite the large number ofnon-immunogenic HLA-ligands identified, all these peptides are naturallypresented on HLA molecules, providing a solid foundation for epitopescreening. By employing in silico analyses only, it would have beennecessary to screen thousands of peptides to identify the inventors' setof epitopes, as opposed to 368 in the inventors' approach.

Infection of various cell cultures expressing distinct HLA alleles withdifferent HCMV deletion mutants will allow for deeper and broaderinsights into the quality of viral CD8+ T-cell targets. Moreover,methods such as ribosome profiling, will enable the identification ofnovel open reading frames that might be a source of T-cell epitopes (46,47). The presence of a broad range of specific memory T cells in healthyseropositive individuals suggests that strategies employing subdominantepitopes and targeting multiple antigens in vaccination and cellulartherapies may be beneficial for sustainable virus control (48-50).Therefore, the identification of a large number of immunogenicHCMV-derived cytotoxic T-cell targets for the most frequent HLArestrictions is, in the inventors' opinion, indispensable for thedevelopment and improvement of such therapies.

In the context of the present invention, the use of HCMV gene deletionmutants lacking various immunoevasins, for the first time enabled thedirect isolation and mass spectrometric identification of roughly 380HCMV-specific HLA-I peptide ligands eluted from twelve different HLAallotypes. Of these peptides 28% induced memory T-cell responses withmultifunctional (IFNy, TNFα, CD107a) effector functions in HCMV-positivedonors. Finally, real-time cytotoxicity assays demonstrated highlyeffective cell lysis of HCMV-infected target cells by peptide-specificCD8+ T-cell clones in vitro.

These results confirm that viral HLA-I ligands eluted from infectedfibroblast cell cultures reflect physiological peptide processing andpresentation mechanisms and are able to induce immunity against HCMV.Therefore, these peptides present novel targets for the treatment ofHCMV-associated pathologies by antigen-specific immunotherapy.

In summary, the present invention presents a novel strategy, whichenables the direct identification of HCMV-derived T-cell epitopes bymass spectrometry. The inventors provide a panel of novel T-cellepitopes and present evidence for their involvement in physiologicalimmune control of HCMV infections. The inventors' study reveals newtargets and provides important insights for the management ofCMV-associated pathologies by antigen-specific immunotherapy.

The present invention will now be described with reference to thefollowing non-limiting examples, nevertheless, without being limitedthereto. For the purposes of the present invention, all references ascited herein are incorporated by reference in their entireties.

FIG. 1 shows that deletion of the genes US2-US11 allows high level ofHLA-I expression. a) MRC-5 or HF-99/7 fibroblasts were mock treated orinfected with AD169VarL wild-type virus or deletion mutants with an MOIof 5. Cell surface expression of HLA-I (W6/32) was analyzed by flowcytometry at 48 h.p.i. b) The rate of infection was determined using thecells from (a). The cells were permeabilized and treated with Fc-FITC,which binds to the HCMV encoded Fc-receptors (vFcR). FITC levels weredetermined by flow cytometry.

FIG. 2 shows the identification of HCMV-derived HLA ligands from MRC-5lung fibroblasts by LC-MS/MS. a) Overview of HLA ligand identificationsobtained by LC-MS/MS analysis of MRC-5 cells after mock treatment (n=2independent experiments), infection with AD169VarL (n=1), infection withthe deletion viruses AD169 ΔUS2-6 (n=3) or ΔUS2-6/ΔUS11 (n=5).Identified ligands of mock treated (n=1) or AD169 ΔUS2-11 infected (n=1)HF-99/7 cells are depicted on the right side. Peptide identificationswere defined as HLA ligands if they showed predicted HLA binding definedas NetMHC IC50≤500 nM and/or normalized SYFPEITHI scores≥50%. The purityof HLA ligand extracts (i.e. the ratio of predicted binders/totalpeptide identifications) of the individual HLA ligand elutions isindicated by red triangles. b) Overlap analysis of the combined datasetsof HCMV individual HLA ligands identified on MRC-5 cells infected withthe three different virus variants. c) Overlap of HCMV-derived HLAligands identified in three independent experiments using MRC-5 cellsinfected with the deletion virus ΔUS2-6. d) Distribution of HLArestrictions among the 198 (MRC-5) and 181 (HF-99/7) unique HCMV-derivedHLA ligands identified in total. Abbreviations: IDs, identifications.

FIG. 3 shows the identification and characterization of naturallypresented T-cell epitopes by ELISpot. HCMV ligands were tested formemory T-cell response by IFNγ ELISpot with PBMCs of healthy,seropositive donors. a) Distribution of dominant and subdominant HCMVligands restricted to HLA-A and -B 19 allotypes. b) Proportion ofepitope source proteins assigned to five different temporal classes ofprotein expression according to Weekes et al. (36). Source proteins notassigned to one of those classes are depicted as not determined (ND). c)ELISpot screening of positively tested HLA-B*07:02-restricted peptides.Shown are numbers of IFNγ spot forming cells (SFC) for each tested donorminus the spot numbers of the negative control of the respective donor.Spot counts of >1000 were set to 1000 because of inaccurate spot countdue to technical limitations. Positive evaluated spot counts aredepicted in black, negative evaluated spot counts in grey. d) Comparisonof IFNγ SFC in ex vivo ELISpots (black) and ELISpots with prior 12 dayamplification (grey). Shown are exemplary results of five donors eachfor two B*08 epitopes (UL34 180-188 and UL26 61-69).

FIG. 4 shows the characterization of HCMV-specific memory T-cells. a)Representative tetramer staining after 12 day amplification in vitro ofCD8+ T cells derived from HLA-matched healthy donors. Shown are resultsof three peptides per HLA. Novel HCMV ligands are compared to known pp65epitopes (left column). b) Exemplary intracellular IFNγ and TNFαstaining of healthy donors' PBMCs after 12 day amplification. Cells werestimulated with novel HCMV peptides or known pp65 epitopes. Barsrepresent percentage of CD8+ T cells producing IFNγ (black), TNFα (grey)or both (light grey). Three peptides per HLA restriction, tested in oneHLA-matched donor, are shown.

FIG. 5 shows the characterization and cytotoxicity of HCMV-specific CD8+T-cell clones. a) Exemplary staining of a UL23 22-30-specific T-cellclone (B*07:02 restricted) with the respective tetramer andintracellular cytokine staining with TNF, IFNγ and the degranulationmarker CD107a. b-d) Real-time cytotoxicity of different UL2322-30-specific T-cell clones monitored by the xCELLigence system. 20,000cells/well of infected or not infected MRC-5 cells were seeded into96-well E-plates. After attachment of target cells, effector cells wereadded 48 h.p.i. at indicated E:T ratios (t0). Synthetic peptides wereadded to target cells one hour prior to effector cells (finalconcentration 1 μg/ml). Impedance was measured every 15 min andnormalized to impedance of wells with medium only. The resultingdimensionless normalized cell index indicates the changes in impedancenormalized to t0. Percentage of lysis was calculated in relation tocells without effector T cells. Experiments were performed intriplicates. b) MRC-5 cells were loaded with specific (UL23 22-30,RPWKPGQRV) (SEQ ID NO: 28) or unspecific (HIV Nef 128-137, TPGPGVRYPL)(SEQ ID NO: 103) peptide or infected with AD169 ΔUS2-6 (MOI 2) andincubated with effector cells in an E:T ratio of 5:1. Controls wereMRC-5 cells without effector cells or without peptide. c) Comparison ofspecific lysis of ΔUS2-US6-infected MRC-5 cells with different E:Tratios. d) Specific lysis of AD169VarL wild type infected orpeptide-loaded cells with indicated E:T ratios.

FIG. 6: The rate of infection was determined using the cells fromFIG. 1. The cells were permeabilized and treated with Fc-FITC, whichbinds to the HCMV encoded Fc-receptors (vFcR). FITC levels weredetermined by flow cytometry.

FIG. 7: Overlap of HCMV-derived HLA ligands between five independent HLAligand elutions from MRC-5 cells infected with AD169 ΔUS2-6/AU1.

FIG. 8: ELISpot screening of positively tested peptides with HLA-A*02:01(a), A*29:02 (b), B*44:02 (c), A*01:01 (d), A*03:01 (e), B*08:01 (f) andB*51:01 (g) restriction. Shown are numbers of IFNγ spot forming cells(SFC) for each tested donor minus the spot numbers of the negativecontrol of the respective donor. Positive evaluated donors are depictedin black, negative tested donors in grey.

FIG. 9: Parallel recognition of multiple HCMV epitopes. ExemplaryELISpot results after 12 day amplification with HLA-B*07-restricted (a)and HLA-B*44-restricted (b) epitopes using PBMCs of two and threedonors, respectively. PBMCs were stimulated with ten novel and alreadyknown epitopes (column 1-10). a) UL83 265-275 (RPHERNGFTVL, column 10)(SEQ ID NO: 102) is a previously identified epitope which was notcontained in the here identified ligands. HIV Nef 128-137 (TPGPGVRYPL)(SEQ ID NO: 103) and medium served as negative controls,Phytohaemagglutinin (PHA) as positive control. b) UL83 364-373(SEHPTFTSQY) (SEQ ID NO: 110) and UL83 511-521 (QEFFWDANDIY) (SEQ ID NO:111) are already known epitopes that were not found as ligands in thisstudy. UL57 193-203 (EEIPASDDVLF) (SEQ ID NO: 107) served as negativecontrol.

FIG. 10: Overview of frequencies of recognition by healthy donors forall identified HCMV epitopes. Dashed line indicates threshold fordominant epitopes.

FIG. 11: Infection of MRC-5 cells with AD169 ΔUS2-6 for followingcytotoxicity testing of peptide-specific T cell clones. a) Comparison ofmorphology of uninfected and infected (20 h.p.i., MOI 1) MRC-5 cells. b)Titration of MOIs in comparison with uninfected (mock) MRC-5 cells forthe xCelligence system. 20,000 cells/well of infected or not infectedMRC-5 cells were seeded into 96-well E-plates. Impedance was measuredevery 15 min and normalized to impedance of wells with medium only. Theresulting dimensionless normalized cell index indicates the changes inimpedance normalized to t0. Experiment was performed in triplicate.

Table 1 shows peptide epitopes of the invention, the source (underlying)protein, sequence, and other data relating to the peptides.

Table 2 shows data for preferred dominant epitopes of the invention.

EXAMPLES Methods Cells and Viruses

MRC-5 fibroblasts (ECACC 05090501) and human foreskin fibroblasts(HF-99/7 ; donated as kind gift by Dieter Neumann-Haefelin and ValeriaKapper-Falcone, Freiburg) were grown in DMEM supplemented with 10% FCS,penicillin and streptomycin. The AD169VarL-based BAC mutants (51) werepropagated on MRC-5 cells.

The recombinant HCMV mutants ΔUS2-6, ΔUS2-6/ΔUS11 and ΔUS2-11 weregenerated according to a previously published procedure (52) using theBAC-cloned AD169varL genome pAD169 (51) as parental BAC. Briefly, a PCRfragment was generated using the primers

KL-DeltaUS11-Kana1 (SEQ ID NO: 115) CAAAAAGTCTGGTGAGTCGTTTCCGAGCGACTCGAGATGCACTCCGCTTCAGTCTATATACCAG TGAATTCGAGCTCGGTAC andKL-DeltaUS11-Kana2 (SEQ ID NO: 116) TAAGACAGCCTTACAGCTTTTGAGTCTAGACAGGGTAACAGCCTTCCCTTGTAAGACAGAGACC ATGATTACGCCAAGCTCCand the plasmid pSLFRTKn (53) as template DNA. The PCR fragmentcontaining a kanamycin resistance gene was inserted 11 into the parentalBAC by homologous recombination in E. coli. Correct mutagenesis wasconfirmed by Southern blot and PCR analysis. Recombinant HCMVs werereconstituted from HCMV BAC DNA by Superfect (Qiagen) transfection intopermissive MRC-5 cells. Virus titers were determined by standard plaqueassay.

Flow Cytometry Analysis of Infected Cells

Cells were detached with accutase (Sigma) and stained with antibodiesdiluted in 3% FCS/PBS. Cells were washed in 3% FCS/PBS supplemented withDAPI and fixed in 3% paraformaldehyde. For intracellular staining ofviral Fc-receptors cells were fixed and permeabilized using the BDCytofix/Cytoperm™ Kit and stained with Fc-FITC (Rockland ImmunochemicalsInc). Cells were measured with a BD FACSCanto™ II system (BDBiosciences) and acquired data was analyzed by FlowJo (v10.1, Tree StarInc.). Analysis of HLA ligands by liquid chromatography-coupled tandemmass spectrometry (LC-MS/MS) Approximately 2-3×10⁸ cells (30 t175flasks) of MRC-5 fibroblasts (A*02:01, A*29:02, B*07:02, B*44:02,C*05:01, and C*07:02) or human foreskin fibroblasts (HF-99/7) (A*01:01,A*03:01, B*08:01, B*51:01, C*01:02, C*07:01) were infected with an MOIof 4-7. At 48 h.p.i., the cells were collected by scraping and washedthree times with PBS. The cell pellet was stored at −80° C. HLA-Iligands were isolated using standard immunoaffinity purificationemploying the pan-HLA class I-specific mAb W6/32 (54). HLA ligandextracts were analyzed as described previously (54). In brief, HLAligand extracts were separated by reversed-phase liquid chromatography(nanoUHPLC, UltiMate 3000RSLCnano, Dionex) using a 75 μm×25 cm PepMapC18 column (Thermo Fisher Scientific). Linear gradients were appliedranging from 2.4% to 32% AcN over the course of 90 min in almost allanalyses. In single experiments other methods, applying 195 or 300 mingradients on a 75 μm×50 cm PepMap column, were tested. Peptides elutedfrom MRC-5 cells were analyzed in an online coupled LTQ Orbitrap XL massspectrometer (Thermo Fisher Scientific) using a top 5 collision inducedfragmentation (CID) method generating ion trap MS/MS spectra. Extractsof HF-99/7 cells were analyzed in an online coupled LTQ Orbitrap FusionLumos mass spectrometer (Thermo Fisher Scientific) using a top speed CIDmethod leading to orbitrap MS/MS spectra. Database search and filteringData processing was performed as described previously (55). In brief,the Mascot search engine (Mascot 2.2.04; Matrix Science) (for ion trapfragment spectra) or the SEQUEST HT search engine (University ofWashington) (for Orbitrap fragment spectra) (56) were used to search thehuman and HCMV proteome. Ion trap spectra were searched against aconcatenated FASTA consisting of the Swiss-Prot reviewed human(September 2013; 20,279 sequences contained) and HCMV proteomes (April2014; 400 sequences contained). Orbitrap spectra were searched against aFASTA consisting of the Swiss-Prot database of reviewed human proteins(March 2016; 20,270 sequences) and the HCMV proteome. The searchcombined data of technical replicates and was not restricted byenzymatic specificity. Precursor mass tolerance was set to 5 ppm, andfragment mass tolerance to 0.5 Da for ion trap spectra analyzed byMascot and 0.02 Da for orbitrap spectra analyzed by SEQUEST HT,respectively. Oxidized methionine was allowed as a dynamic modification.FDR was estimated using the Percolator algorithm (57).

Peptide-12 spectrum matches were filtered for an FDR of 5%, searchengine rank=1 and peptide lengths of 8-12 aa. For Mascot databasesearches the additional filter of Mascot Ion Scores≥20 were utilized.Peptide identifications were annotated to their respective HLA motifsusing both SYFPEITHI (37), with a normalized score of ≥50%, andNetMHCv3.4 (58) for MRC-5 or Net MHCpan3.0 (59) for HF-99/7, applying IC50≤500 nM percentile rank<2% (for NetMHCpan3.0) as cutoffs. Peptidesfulfilling the cutoff in either or both prediction tools were designatedas HLA ligands in this manuscript. In case of multiple possibleannotations, the HLA allotype yielding the best rank/score was selected.Peptides were tested in donor samples of different restrict ions if thetwo algorithms resulted in inconsistent allotype annotations. Peptideand HLA: peptide monomer synthesis Synthetic peptides were produced bystandard 9-fluorenylmethyloxycarbonyl/tert-butyl strategy using peptidesynthesizers 433A (Applied Biosystems, Darmstadt, Germany), P11(Activotec, Cambridge, UK) or Liberty Blue (CEM, Kamp-Lintfort,Germany). Purity was assessed by reversed phase HPLC (e2695, Waters,Eschborn, Germany) and identity affirmed by nano-UHPLC (UltiMate 3000RSLCnano) coupled online to a hybrid mass spectrometer (LTQ Orbitrap XL,both Thermo Fisher). Lyophilized peptides were dissolved at 10 mg/ml inDMSO and diluted 1:10 in bidestilled H₂O. Frozen aliquots were furtherdiluted in cell culture medium and sterile filtered if necessary.Synthetic peptides were used for validation of LC-MS/MS identificationsas well as for functional experiments. Biotinylated recombinant HLAmolecules and fluorescent HLA:peptide tetramers were produced asdescribed previously (60-62). Target cell infection for cytotoxicityassays MRC-5 cells were cultured in DMEM (1×) (Life technologies)supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/m1streptomycin at 37° C. and 7.5% CO₂. For cytotoxicity assays MRC-5 cellswere infected with an MOI of 2 and subsequently centrifuged for 30 minat 300 g. After resting for approximately 1 h cells were harvested bytrypsination for 2 min at 37° C. and seeded in E-plates 96 (Roche) with20,000 cells per well. T-cell culture blood samples were kindly providedby the Institute for Clinical and Experimental Transfusion Medicine atthe University Hospital of Tubingen after obtaining written informedconsent. Peripheral blood mononuclear cells (PBMCs) were isolated fromhealthy HCMV-seropositive blood donors by Ficoll-Hypaque densitygradient centrifugation. Cells were frozen at −80° C. in FCS+10% DMSO.After thawing, cells were rested overnight prior to stimulation. Cultureconditions were 7.5% CO₂ and 37° C. in humidified incubators in IMDM(PAA) supplemented with 5% heat-inactivated pooled human plasma(isolated from healthy blood donors), 100 U/ml penicillin, 100 μg/mlstreptomycin, 25 μg/ml gentamicin (Life technologies) and 50 μMβ-mercaptoethanol (Carl Roth). IFNγ-ELISpot assay The IFNγ-ELISpot assaywas performed after 12 day stimulation as described previously (62) ordirectly ex vivo one day after thawing. Readout was performed accordingto manufacturers' recommendation and the cancer immunotherapy monitoringpanel (63). PHA was used as positive control. The following 13 peptides,restricted to the respective HL A, served as negative controls:GSEELRSLY HIV POL 71-79 (A*01) (SEQ ID NO: 114), YLLPAIVHI HUMAN DDX5148-156 (A*02) (SEQ ID NO: 104), RLRPGGKKK HIV GAG 20-28 (A*03) (SEQ IDNO: 105), TPGPGVRYPL HIV Nef 128-137 (B *07) (SEQ ID NO: 113), GGKKKYKLHIV GAG 24-31 (B*08) (SEQ ID NO: 106), EEIPASDDVLF HCMV DNBI 1095-1105(B*44) (SEQ ID NO: 107), DPYKATSAV HUMAN MUC16 6326-6334 (B *51) (SEQ IDNO: 108). DMSO was used as a negative control for HLA-A*29. Blue spotsspecific for IFNγ-producing cells were automatically counted using anImmunoSpot S5 analyzer (CTL) and ImmunoSpot Software. T-cell responseswere considered to be positive when >10 spots/well were counted and meanspot count per well was at least 3-fold higher than the mean number ofspots in negative control wells. Background staining due to excesscytokine and overlapping spots hamper the detection of reliable countsin wells of highly responsive donors. Therefore, spot counts of >1000 or“too numerous to count” were set to 1000. Analysis of T cells HLAtetramer staining of T cells was performed by incubation with 5 μg/mltetramer diluted in tetramer staining buffer (2% FCS, 0.01% sodium azideand 2 mM EDTA in PBS) for 30 min at 4° C. Afterwards, T cells werestained with CD8-PerCP (Biolegend) for 20 min at 4° C. For ICS 0.5-1 Miocells/well were stimulated with individual peptides (10 μg/ml) inpresence of BrefeldinA (Sigma-Aldrich), GolgiStop (BD Biosciences) andanti-CD107a-FITC mAB (BD Biosciences) in 150 μl per well for 12-14 h.After incubation cells were washed and stained with anti-CD8-PerCP(Biolegend) and anti-CD4-APC (BD Biosciences) followed by fixation andpermeabilization for further 20 min at 4° C. (Cytofix/Cytoperm, BDBiosciences). After washing with permwash buffer cytokines were stainedwith anti-TNFα-PacificBlue (Biologend) and anti-IFNγ-PE (BD Biosciences)for 20 min at 4° C. Flow cytometric measurements were performed on aFACSCanto II cytometer (BD Biosciences) with the DIVA software andanalyzed using FlowJo Version 10. T-cell clones PBMCs of HLA-matchedseropositive donors were stimulated with 1 μg/ml specific peptide oneday after thawing and IL-2 (20 U/ml) (Novartis) on day 2 and 5. On day14 HLA tetramer staining was performed and tetramer-positive CD8+ Tcells were sorted in 96-well plates containing 1.5×10⁵ irradiated PBMCs(60-Gray, 1000 Elite Gammacell), 1.5×10⁴ irradiated LG2-EBV (200 Gray)(kind gift of Pierre van der Bruggen, Ludwig Institute for CancerResearch, Brussels, Belgium) as feeder cells, 150 U/ml IL-2 and 0.5μg/ml PHA-L (Sigma-Aldrich) in 150 μl media per well. Sorting wasperformed using BD FACSJazz™ equipped with BD FACS™ Software. Five orten tetramer-positive CD8+ T cells were sorted per well and incubated at37° C. and 7.5% CO₂. After resting for one week cells were stimulatedtwice per week with 150 U/ml IL-2, freshly irradiated feeder cells (asdescribed above) were added every second or third week together with 150U/ml IL-2 and 1m/ml PHA-L (Roche).

Real-Time Cytotoxicity Assay (XCelligence)

Real-time cytotoxicity assays were carried out as described previously(64). All experiments were performed in DMEM with 10% FCS and 1%PenStrep. Background values were determined using 50 μl medium per well.MRC-5 cells, infected or uninfected, were seeded in 96-well E-plates(Roche) at a concentration of 20,000 cells per well in 50 μl medium.Effector cells were added 48 h after target cells in 14 indicated E:Tratios. In case of peptide loading of MRC-5 cells, synthetic peptides(f.c. 1 μg/ml) were added to target cells one hour prior to effectorcells. Cell attachment was monitored using the RTCA SP (Roche)instrument and the RTCA software Version 1.1 (Roche). Impedancemeasurements were performed every 15 minutes for up to 140 h. Allexperiments were performed in triplicates.

Results

The epitope peptides as well as their characteristics as determined aredepicted in the following tables 1 and 2:

TABLE 1 Peptide epitopes of the invention, the source (underlying)protein, sequence, and other data relating to the peptides Actual HLArestric- tion % Posi- (identi- tive fied donors % Pre- using Posi-(after Posi- Intra- Source dicted tetramer tive 12 tive cellu- proteinHLA staining donors days of donors lar Cell and Sequence/ restric- andas stimu- (ex cytokine Tetramer- line position SEQ ID NO: tion ICS)tested lation) vivo) staining staining MRC- US8 74-82 GVLDAVWRV A*02:01A*02:01 13/18 72.2 50.0 CD8 Positive 5 (SEQ ID NO: 1) MRC- UL150A 152-ALWDVALLEV A*02:01 A*02:01 10/14 71.4 25.0 CD8 Positive 5 161(SEQ ID NO: 2) MRC- UL100 200- TLIVNLVEV A*02:01 A*02:01 11/20 55.0 25.0CD8 Positive 5 208 (SEQ ID NO: 3) MRC- UL44 259- GLFAVENFL A*02:01 DR 7/13 53.9 0.0 CD4 Nt 5 267 (SEQ ID NO: 4) MRC- UL71 40-48 FLDENFKQLA*02:01 DR 10/21 47.6 37.5 CD4 Negative 5 (SEQ ID NO: 5) MRC- UL105 431-RLFDLPVYC A*02:01  5/12 41.7 Nt Nt Nt 5 439 (SEQ ID NO: 6) MRC-UL29 175- RLQPNVPLV A*02:01  6/18 33.3 0.0 Nt Nt 5 183 (SEQ ID NO: 7)MRC- US16 134- GLLAHIPALGV A*02:01  6/22 27.3 0.0 Nt Nt 5 144(SEQ ID NO: 8) MRC- US29 293- ALSPSTSKV A*02:01  4/17 23.5 Nt Nt Nt 5301 (SEQ ID NO: 9) MRC- UL29 344- SLYEANPEL A*02:01  4/17 23.5 Nt Nt Nt5 352 (SEQ ID NO: 10) MRC- UL86 146- TILDKILNV A*02:01  4/20 20.0 Nt NtNt 5 154 (SEQIDNO: 11) MRC- US16 186- TLINGVWVV A*02:01  2/11 18.2 Nt NtNt 5 194 (SEQ ID NO: 12) MRC- US16 134- GLLAHIPAL A*02:01  2/11 18.2 NtNt Nt 5 142 (SEQ ID NO: 13) MRC- UL48 132- ALYPEYIYTV A*02:01  3/18 16.7Nt Nt Nt 5 141 (SEQ ID NO: 14) MRC- UL47 766- GLNERLLSV A*02:01  3/2015.0 Nt Nt Nt 5 744 (SEQ ID NO: 15) MRC- UL34 BO- ALFNQLVFTA A*02:01 2/15 13.3 Nt Nt Nt 5 138 (SEQ ID NO: 16) MRC- UL56 124- FTDNVRFSVA*02:01  1/13 7.8 Nt Nt Nt 5 132 (SEQ ID NO: 17) MRC- UL128 145-GLDQYLESV A*02:01  1/13 7.8 Nt Nt Nt 5 153 (SEQ ID NO: 18) MRC-UL84 133- ALLGRLYFI A*02:01  1/15 6.7 Nt Nt Nt 5 141 (SEQ ID NO: 19)MRC- UL4 96-104 NYNEQHYRY A*29:02  5/13 38.5 Nt Nt Nt 5 (SEQ ID NO: 20)MRC- US27 276- LYVGQFLAY A*29:02  3/12 25.0 Nt Nt Nt 5 284(SEQ ID NO: 21) MRC- UL4 88-97 SFFPKLQGNY A*29:02  2/9 22.2 Nt Nt Nt 5(SEQ ID NO: 22) MRC- UL4 89-97 FFPKLQGNY A*29:02  2/12 16.7 Nt Nt Nt 5(SEQ ID NO: 23) MRC- UL16 162- YPRPPGSGL B*07:02 B*07:02 19/22 86.4 25.0Nega- Positive 5 170 (SEQ ID NO: 24) tive MRC- UL83 417- TPRVTGGGAMB*07:02 B*07:02 31/38 81.6 100.0 CD8 Positive 5 426 (SEQ ID NO: 25) MRC-TRS1 166- SPRDAWIVL B*07:02 B*07:02 15/22 68.2 20.0 CD8 Positive 5 174(SEQ ID NO: 26) MRC- UL52 349- SPSRDRFVQL B*07:02 B*07:02 14/21 66.733.3 Nega- Positive 5 357 (SEQ ID NO: 27) tive MRC- UL23 22-30 RPWKPGQRVB*07:02 B*07:02 15/28 53.6 66.7 CD8 Positive 5 (SEQ ID NO: 28) MRC-UL46 76-84 SPRHLYISL B*07:02 B*07:02 11/22 50.0 0.0 CD4/CD8 Positive 5(SEQ ID NO: 29) MRC- UL38 225- IPMTFVDRDSL B*07:02  5/14 35.7 Nt Nt Nt 5235 (SEQ ID NO: 30) MRC- US30 313- RPFPSTHQL B*07:02  4/13 30.8 0.0 NtNt 5 321 (SEQ ID NO: 31) MRC- UL83 49-57 RVSQPSLIL B*07:02  4/15 26.7 NtNt Nt 5 (SEQ ID NO: 32) MRC- UL82 245- SPHPPTSVFL B*07:02  3/12 25.0 NtNt Nt 5 254 (SEQ ID NO: 33) MRC- UL27 485- IPDYRSVSL B*07:02  4/18 22.2Nt Nt Nt 5 493 (SEQ ID NO: 34) MRC- UL31 310- APFGRVSV B*07:02  3/1520.0 Nt Nt Nt 5 317 (SEQ ID NO: 35) MRC- TRS1/IRS1 IPVERQAL B*07:02 2/12 16.7 Nt Nt Nt 5 92-99 (SEQ ID NO: 36) MRC- UL98 135- APNYRQVELB*07:02  2/12 16.7 Nt Nt Nt 5 143 (SEQ ID NO: 37) MRC- UL40 210-LPNDHHYAL B*07:02  1/17 5.9 Nt Nt Nt 5 218 (SEQ ID NO: 38) MRC-US12 82-89 APYLRDTL B*07:02  1/19 5.3 Nt Nt Nt 5 (SEQ ID NO: 39) MRC-UL112/UL11 SENGNLQVTY B*44:02 B*44:02 26/31 83.9 62.5 CD8 Positive 53 125-134 (SEQ ID NO: 40) MRC- ULI 17 358- HETGVYQMW B*44:02 B*44:0217/26 65.4 62.5 CD8 Positive 5 366 (SEQ ID NO: 41) MRC- UL17 24-32DEQVSKRSW B*44:02 B*44:02 11/24 45.8 12.5 CD8 Positive 5 (SEQ ID NO: 42)MRC- TRL12 402- SESEFIVRY B*44:02 B*44:02  8/20 40.0 37.5 CD8 Positive 5410 (SEQ ID NO: 43) MRC- UL147A51- EEQDYRALL B*44:02  4/12 33.3 Nt Nt Nt5 59 (SEQ ID NO: 44) MRC- UL78 ISO- RENAGVALY B*44:02  4/21 19.1 Nt NtNt 5 158 (SEQ ID NO: 45) MRC- US21 71-80 AEPNFPKNVW B*44:02  2/14 14.3Nt Nt Nt 5 (SEQ ID NO: 46) MRC- TRSl/IRS1 EEATALGREL B*44:02  1/10 10.0Nt Nt Nt 5 276-285 (SEQ ID NO: 47) MRC- US 11 103- SESLVAKRY B*44:02 1/10 10.0 Nt Nt Nt 5 111 (SEQ ID NO: 48) MRC- UL54 755- LENGVTHRFB*44:02  1/14 7.1 Nt Nt Nt 5 763 (SEQ ID NO: 49) MRC- US22 72-81REQAAIPQIY B*44:02  1/16 6.3 Nt Nt Nt 5 (SEQ ID NO: 50) HF- UL105 715-YADPFFLKY A*01:01 A*01:01 15/15 100.0 90.9 CD8 Positive 99/7 723(SEQ ID NO: 51) HF- UL44 245- VTEHDTLLY A*01:01 A*01:01 13/14 92.9 100.0Nt Nt 99/7 253 (SEQ ID NO: 52) HF- UL69 569- RTDPATLTAY A*01:01 A*01:0119/23 82.6 66.7 CD8 Positive 99/7 578 (SEQ ID NO: 53) HF- US28 122-ITEIALDRY A*01:01 A*01:01 14/24 58.3 14.3 CD8 Positive 99/7 130(SEQ ID NO: 54) HF- UL55 657- NTDFRVLELY A*01:01 A*01:01  9/16 56.3 0.0CD8 Positive 99/7 665 (SEQ ID NO: 55) HF- UL36 82-91 FVEGPGFMRY A*01:01 5/14 35.7 Nt Nt Nt 99/7 (SEQ ID NO: 56) HF- UL148 282- SLDRFIVQYA*01:01 DR  5/14 35.7 Nt CD4 Nt 99/7 290 (SEQ ID NO: 57) HF- UL25 370-YTSRGALYLY A*01:01  3/14 21.4 Nt Nt Nt 99/7 379 (SEQ ID NO: 58) HF-UL86 1346- TSETHFGNY A*01:01  3/15 20.0 Nt Nt Nt 99/7 1354(SEQ ID NO: 59) HF- US34 92-101 GSDALPAGLY A*01:01  3/16 18.8 Nt Nt Nt99/7 (SEQ ID NO: 60) HF- UL48 1607- VTDYGNVAFK A*01:01  3/16 18.8 Nt NtNt 99/7 1617 Y (SEQ ID NO: 61) HF- IRSl/TRSI LLDELGAVFG A*01:01  2/1315.4 Nt Nt Nt 99/7 464-474 Y (SEQ ID NO: 62) HF- UL112/UL113 ISENGNLQVTYA*01:01  3/20 15.0 Nt Nt Nt 99/7 124-134 (SEQ ID NO: 63) HF- UL105 616-VTDPEHLMM A*01:01  2/14 14.3 Nt Nt Nt 99/7 624 (SEQ ID NO: 64) HF-UL105 360- DLDFGDLLKY A*01:01  2/16 12.5 Nt Nt Nt 99/7 369(SEQ ID NO: 65) HF- UL78 222- YSDRRDHVWS A*01:01  1/16 6.3 Nt Nt Nt 99/7232 Y (SEQ ID NO: 66) HF- UL77 228- GLYTQPRWK A*03:01 A*03:01 16/21 76.250.0 CD8 Positive 99/7 236 (SEQ ID NO: 67) HF- UL57 790- RVKNRPIYRA*03:01 A*03:01 14/23 60.9 33.3 CD8 Positive 99/7 798 (SEQ ID NO: 68)HF- UL36 51-60 RSALGPFVGK A*03:01 A*03:01  6/15 40.0 Nt CD8 Positive99/7 (SEQ ID NO: 69) HF- UL123 184- KLGGALQAK A*03:01  6/15 40.0 Nt NtNt 99/7 192 (SEQ ID NO: 70) HF- US33A 13-21 KLGYRPHAK A*03:01 A*03:0111/29 37.9 Nt CD8 Positive 99/7 (SEQ ID NO: 71) HF- US24 136- RVYAYDTREKA*03:01  4/11 36.4 Nt Nt Nt 99/7 145 (SEQ ID NO: 72) HF- UL25 580-GVSSVTLLK A*03:01  5/14 35.7 Nt Nt Nt 99/7 588 (SEQ ID NO: 73) HF-UL84 3-11 RVDPNLRNR A*03:01  5/15 33.3 Nt Nt Nt 99/7 (SEQ ID NO: 74) HF-UL70 698- SVRLPYMYK A*03:01  4/16 25.0 Nt Nt Nt 99/7 706 (SEQ ID NO: 75)HF- UL79 237- RTFAGTLSR A*03:01  3/14 21.4 Nt Nt Nt 99/7 245(SEQ ID NO: 76) HF- UL57 1044- RLADVLIKR A*03:01  2/13 15.4 Nt Nt Nt99/7 1052 (SEQ ID NO: 77) HF- UL70 697- RSVRLPYMYK A*03:01  2/15 13.3 NtNt Nt 99/7 706 (SEQ ID NO: 78) HF- UL122 113- SVSSAPLNK A*03:01  1/147.1 Nt Nt Nt 99/7 121 (SEQ ID NO: 79) HF- UL13 465- YLVRRPMTI B*08:01B*08:01 11/22 50.0 33.3 Nega- Positive 99/7 473 (SEQ ID NO: 80) tive HF-UL36 199- VMKFKETSF B*08:01  5/13 38.5 Nt Nt Nt 99/7 207 (SEQ ID NO: 81)HF- UL84 239- TPLLKRLPL B*08:01  4/14 28.6 Nt Nt Nt 99/7 247(SEQ ID NO: 82) HF- UL40 170- HLKLRPATF B*08:01  3/13 23.1 Nt Nt Nt 99/7178 (SEQ ID NO: 83) HF- UL84 500- FISSKHTL B*08:01  3/14 21.4 Nt Nt Nt99/7 507 (SEQ ID NO: 84) HF- UL44 26-34 QLRSVIRAL B*08:01  2/14 14.3 NtNt Nt 99/7 (SEQ ID NO: 85) HF- UL148 1-8 MLRLLFTL B*08:01 B*08:01  1/147.1 Nt Nt Nt 99/7 (SEQ ID NO: 86) HF- UL83 116- LPLKMLNI B*51:01 B*51:0112/15 80.0 87.5 CD8 Positive 99/7 123 (SEQ ID NO: 87) HF- UL38 156-FPVEVRSHV B*51:01 B*51:01 15/23 65.2 0.0 CD8 Positive 99/7 164(SEQ ID NO: 88) HF- UL56 503- DARSRIHNV B*51:01 B*51:01  8/15 53.3 NtCD8 Positive 99/7 511 (SEQ ID NO: 89) HF- UL71 330- IPPPQIPFV B*51:01 6/15 40.0 Nt Nt Nt 99/7 338 (SEQ ID NO: 90) HF- US28 158- IAIPHFMVVB*51:01  5/15 33.3 Nt Nt Nt 99/7 166 (SEQ ID NO: 91) HF- US23 65-73IPHNWFLQV B*51:01  5/15 33.3 Nt Nt Nt 99/7 (SEQ ID NO: 92) HF- UL33 162-VPAAVYTTV B*51:01  5/15 33.3 Nt Nt Nt 99/7 170 (SEQ ID NO: 93) HF-ULM 66-74 FPAHDWPEV B*51:01  2/15 13.3 Nt Nt Nt 99/7 (SEQ ID NO: 94) HF-UL122 449- MPVTHPPEV B*51:01  2/15 13.3 Nt Nt Nt 99/7 457(SEQ ID NO: 95) HF- UL75 540- FPDATVPATV B*51:01  1/15 6.7 Nt Nt Nt 99/7549 (SEQ ID NO: 96) HF- UL48 1322- LPYLSAERTV B*51:01  1/15 6.7 Nt Nt Nt99/7 1331 (SEQ ID NO: 97) MRC- UL147A 2-10 SLFYRAVAL A*02:01  5/13 38.512.5 Nt Nt 5 (SEQ ID NO: 98) B*08:01  4/22 18.2 Nt Nt Nt HF- 99/7 HF-UL26 61-69 LPYPRGYTL B*08:01 B*08:01/ 11/16 68.8 16.7 CD8 positive 99/7(SEQ ID NO: 99) B*51:01 B51*:01 10/16 62.5 33.3 CD8 positive HF-B*08:01/ 99/7 B*51:01 HF- UL34 180- LPHERHREL B*08:01 B*08:01 20/22 90.985.7 CD8 positive 99/7 188 (SEQ ID NO: B*07:02  3/12 25.0 Nt Nt Nt MRC-100) 5

TABLE 2Summary of dominant epitopes. Ex vivo ELISpots were performed using donorsthat were positively tested in ELISpots with 12d stimulation.Abbreviations: 12d stim, 12-day amplification with IL-2 in vitro; nt, not tested.ELISpot actual response ELISpot Intra- HLA rate response cellu- SequenceTested restric- (12 d rate lar Tetramer Protein SEQ ID NO: HLA tionstim.) (ex vivo)† staining staining UL83 495-503 NLVPMVATV* A*02:01A*02:01 75.0 100.0 CD8 positive SEQ ID NO: 101 US8 74-82 GVLDAVWRVA*02:01 A*02:01 72.2 50.0 CD8 positive SEQ ID NO: 1 UL150A 152-ALWDVALLEV A*02:01 A*02:01 71.4 25.0 CD8 positive 161 SEQ ID NO: 2UL100 200-208 TLIVNLVEV A*02:01 A*02:01 55.0 25.0 CD8 positiveSEQ ID NO: 3 UL44 259-267 GLFA VENFL A*02:01 Class II 53.9 0.0 CD4not tested SEQ ID NO: 4 UL16 162-170 YPRPPGSGL* B*07:02 B*07:02 86.425.0 nega- positive SEQ ID NO: 24 tive UL83 417-426 TPRVTGGGAM* B*07:02B*07:02 81.6 100.0 CD8 positive SEQ ID NO: 25 TRS1 166-174 SPRDAWIVLB*07:02 B*07:02 68.2 20.0 CD8 positive SEQ ID NO: 26 UL52 349-357SPSRDRFVQL B*07:02 B*07:02 66.7 33.3 nega- positive SEQ ID NO: 27 tiveUL23 22-30 RPWKPGQRV B*07:02 B*07:02 53.6 66.7 CD8 positiveSEQ ID NO: 28 UL46 76-84 SPRHLYISL B*07:02 B*07:02 50.0 0.0 CD4/positive SEQ ID NO: 29 CD8 UL112/UL113 SENGNLQVTY B*44:02 B*44:02 83.962.5 CD8 positive 125-134 SEQ ID NO: 40 UL117 358-366 HETGVYQMW B*44:02B*44:02 65.4 62.5 CD8 positive SEQ ID NO: 41 UL105 715-723 YADPFFLKY*A*01:01 A*01:01 100.0 90.9 CD8 positive SEQ ID NO: 51 UL44 245-253VTEHDTLLY* A*01:01 A*01:01 92.9 100.0 CD8 positive SEQ ID NO: 52UL69 569-578 RTDPATLTAY A*01:01 A*01:01 82.6 66.7 CD8 positiveSEQ ID NO: 53 US28 122-130 ITEIALDRY A*01:01 A*01:01 58.3 14.3 CD8positive SEQ ID NO: 54 UL55 657-665 NTDFRVLELY A*01:01 A*01:01 56.3 0.0CD8 positive SEQ ID NO: 55 UL77 228-236 GLYTQPRWK A*03:01 A*03:01 76.250.0 CD8 positive SEQ ID NO: 67 UL57 790-798 RVKNRPIYR A*03:01 A*03:0160.9 33.3 CD8 positive SEQ ID NO: 68 UL34 180-188 LPHERHREL B*08:01B*08:01 90.9 85.7 CD8 positive SEQ ID NO: 100 UL26 61-69 LPYPRGYTLB*08:01 B*08:01/ 68.8 16.7 CD8 positive SEQ ID NO: 99 51:01 UL13 465-473YLVRRPMTI B*08:01 B*08:01 50.0 33.3 nega- positive SEQ ID NO: 80 tiveUL83 116-123 LPLKMLNI* B*51:01 B*51:01 80.0 87.5 CD8 positiveSEQ ID NO: 87 UL38 156-164 FPVEVRSHV B*51:01 B*51:01 65.2 0.0 CD8positive SEQ ID NO: 88 UL26 61-69 LPYPRGYTL B*51:01 B*08:01/ 62.5 33.3CD8 positive SEQ ID NO: 99 51:01 UL56 503-511 DARSRIHNV B*51:01 B*51:0153.3 20.0 CD8 positive SEQ ID NO: 89

Deletion of HCMV encoded immunoevasins rescues HLA-I expression ofinfected cells So far, attempts to isolate naturally presented HCMVderived HLA-I ligands have not been successful. HCMV encodes for severalimmunoevasins targeting HLA-I at various stages of the antigenpresentation pathway. Therefore, the inventors speculated that deletionof genes involved in HLA-I regulation would enable the identification ofvirally encoded HLA-I ligands. The inventors constructed AD169VarL (withpartial ULb′ region (35)) deletion mutants lacking the genes US2-6(ΔUS2-6), US2-6+US11 (ΔUS2-6/US11) and US2-11 (ΔUS2-11). To measure thelevel of HLA-I rescue due to lack of specific immunoevasins, theinventors infected two different fibroblast cell cultures expressingHLA-I types of interest: MRC-5 (HLA-A*02:01, -A*29:02, -B*07:02,-B*44:02, -C*05:01, and -C*07:02) and HFF-99/7 (HLA-A*01:01, A*03:01,B*08:01, B*51:01, C*01:02, and C*07:01). The rate of infection wasdetermined using Fc-FITC, which binds to the HC MV encoded Fc-receptors(vFcR) (FIG. 6). At 48 h post-infection (h.p.i.) the HLA-I cell surfacelevel was determined by flow cytometry using the pan-HLA-I antibodyW6/32 (FIG. 1). Interestingly, HLA-I downregulation by AD 169VarLwild-type virus varied strongly between fibroblasts. Since in MRC-5cells HLA-B*44:02 is expressed at very low level in mock treated cells,but is induced strongly in HCMV infected cells, this molecule could bethe reason for the apparent low level of reduction by AD169VarL(compared to mock treated cells). As expected, infection with HCMVmutant viruses lacking HLA-I immunoevasins showed a robust rescue ofHLA-I at the cell surface and the inventors next proceeded with detailedHLA-I ligandome analysis using the virus mutants. Direct identificationof HCMV-derived HLA-I ligands by LC-MS/MS First, for directidentification of processed and presented HCMV-derived HLA-I ligands theinventors performed mass spectrometric analysis ofimmunoaffinity-purified peptide extracts isolated from MRC-5 cells. At48 h.p.i. , HLA-I ligands isolated from cells infected with AD169VarL(n=1 sample), ΔUS2-6 (n=3 samples), ΔUS2-6/ΔUS11 (n=5 samples), and mockcontrols (n=1 sample) were exhaustively analyzed in five to sevenLC-MS/MS runs per sample. These MS analyses revealed 816 to 2,714 uniqueHLA ligands per sample (FIG. 2a ). As expected, only 3/816 (0.4%) of HLAligands, eluted from MRC-5 cells infected with AD169VarL wild-typevirus, were derived from HCMV, while infection with the deletion virusesresulted in substantially increased viral peptide identification ratesand numbers. In MRC-5 cells infected with the mutant viruses ΔUS2-6 andΔUS2-6/ΔUS11 a total of 79 and 181 HCMV-derived HLA ligands wereidentified, respectively, resulting in a total number of 194 uniqueviral peptides. Overlap analysis revealed 66/194 viral peptides to bepresented on MRC-5 after infection with both deletion viruses (FIG. 2b). Interestingly, 13/79 (17%) and 114/181 (63%) viral peptides of ΔUS2-6and ΔUS2-6/ΔUS11 infected cells, respectively, were unique. Thisdemonstrates that the HLA-I immunoevasins not only affect the quantity,but also the quality of HLA-I antigen processing and presentation.Therefore, the use of varying HCMV deletion mutants can result in ahigher variability of identified HCMV-derived peptide species.Furthermore, the inventors isolated the HLA-presented peptides fromseveral biological replicates for each infection to maximize the numberof identified HCMV-derived peptides. Thereby, the inventors were able toidentify between 37 and 63 (mean: 51) unique viral HLA ligands on cellsinfected with ΔUS2-6, corresponding to 2.4-3.0% (mean: 2.8%) of totalHLA ligand identifications. Overlap analysis of viral ligands identifiedin the three independent HLA precipitations revealed 31/79 (39%) ofpeptides to be uniquely identified in a single experiment, while 61%showed reproducible identification in at least two out of threeexperiments (FIG. 2c ). On cells infected with ΔUS2-6/ΔUS11 even higherproportions of viral ligands were identified, resulting in 79-119 (mean:93) unique viral HLA ligands corresponding to 3.2-4.5% (mean: 3.9%) oftotal HLA ligands. Here, a similar degree of reproducibility wasobserved for the five independent precipitations, which resulted in120/181 (66%) reproducible viral ligands (in≥2/5 experiments), while61/181 (34%) were uniquely identified in individual experiments (FIG.7). In total, analyses of infected MRC-5 fibroblasts allowed theidentification of 198 unique HCMV-derived HLA ligands, of which 78, 15,66, 31, 3, and 5 are restricted to HLA-A*02:01, -A*29:02, -B*07:02,-B*44:02, -C*05:01, and -C*07:02, respectively (FIG. 2d ). Due to theapplied 5% false discovery rate (FDR) in data processing, the identified7/2, (0.34%) and 3/1, (0.27%) viral peptides in mock controls (FIG. 2a )are most likely false-positive annotations. In order to estimate theactual false discovery rate of HCMV-derived peptides, the inventorscompared fragment spectra of 50 randomly selected A*02:01 andB*44:02-restricted synthetic peptides to their natural counterparts.Fragmentation patterns matched for 48/50 (96%) spectrum pairs by manualvalidation, which indicates an overall false-positive annotation rate ofHCMV-derived HLA ligands of <5%. To extend the set of HCMV-derivedligands to additional HLA allotypes, the inventors next infected primaryhuman foreskin fibroblasts (HF-99/7) with the ΔUS2-US11 deletion mutant.Peptide extracts from mock treated and infected cells (one sample each)were analyzed in three LC-MS/MS runs yielding a total number of 2,839and 5,511 HLA ligands, respectively (FIG. 2a ). Of these, 37, 44, 21,43, 17, and 4 viral peptides (altogether 181) were restricted toHLA-A*01:01, -A*03:01, -B*08:01, -B*51:01, -C*01:02, and -C*07:01,respectively (FIG. 2d ). The HLA annotation of 15 peptides wasambiguous. Therefore, in total, 368 unique viral HLA-I ligands wereidentified from two different fibroblast cell cultures. Eleven ligandswere found on both cell lines. The inventors had speculated that aninfection time of 48 hrs would allow the detection of peptidesoriginating from proteins with various expression kinetics (36). Indeed,the source proteins of the identified ligands represent all classes ofgene expression. IFNγ ELISpot screening validates numerous HCMV-derivedligands to be T-cell epitopes.

All viral ligands identified from MRC-5 cells and the top ranked ligandsfrom HF-99/7 cells (≥70% SYFPEITHI score, ≤50 nM IC50 and/or <0.5%NetMHC percentile rank) were further tested for immunogenicity. Allpeptides were synthesized in house and tested for memory T-cellresponses in at least seven different HCMV seropositive HLA-matchedindividuals by IFNγ ELISpot assay. HLA restriction and virus-specificityof these T-cell responses was confirmed using HLA mismatched and HCMVseronegative donors as controls. In total, 28% of all peptides weretested positive in at least one individual. This percentage was roughlythe same across all HLA restrictions (FIG. 3a ). Although the inventorsperformed the ligandome analysis at only one time point (48 h.p.i.), alltemporal classes of gene expression (36) were present among the sourceproteins of the identified epitopes (FIG. 3b ). As expected, normalizedspot counts of IFNγ ELISpots after peptide stimulation were donordependent and in part highly variable (FIGS. 3c , 8, and 9). Dependenton the frequency of recognition the inventors grouped the peptides in tothree categories: negative (no memory response in any individual),subdominant (recognized by <50% of individuals) and dominant (recognizedby ≥50% of individuals). Interestingly, in addition to the well-knownepitopes derived from pp65, the inventors found a number of other highlyimmunogenic peptides for each HLA restriction. Most immunogenic peptidesin proportion to the number of tested peptides were found forHLA-A*01:01, whereas the highest percentage of dominant epitopes wasfound for HLA-B*07:02 (FIG. 3a ). The inventors observed that forpeptides with higher recognition rates the mean number of specificmemory T cells after 12 day stimulation is often higher compared topeptides with lower recognition rates (FIGS. 3c and 8). To exclude thatthis effect is caused by competitive effects among the differentepitopes during the 12 day amplification, the inventors additionallyperformed ex vivo IFNγ ELISpots without this prestimulation. Thedominant epitopes were retested with PBMC samples of previously testedpositive donors. Only a few of the best epitopes elicited frequent,detectable responses ex vivo (Table 1). In most cases, memory T-cellnumbers were too small to be detectable ex vivo but underwent, in partmassive, amplification (up to 1000-fold) upon pre-stimulation (FIG. 3d). The amplification rate was highly individual for epitopes as well asfor donors. In total, 103 HCMV-derived T-cell epitopes were identified,whereof 26 were shown to be dominant (Table 2). In case of positiveresults in HLA-I mismatched ELISpots, the respective peptide was testedfor the next best predicted HLA-I allele in ELISpots and/or forCD8+/CD4+ T-cell responses by intracellular cytokine staining (ICS).Three peptides elicited responses by CD4+ T cells, indicating binding toHLA class II. Three epitopes (UL147A 2-10, UL34 180-188, and UL26 61-69)are potentially able to bind to more than one HLA-I allotype since theystimulated T cells of different donors harboring either of two wellpredicted alleles. In summary, in addition to seven previously describedepitopes, the inventors were able to identify 96 novel HCMV-derivedT-cell epitopes. As the inventors have observed a long time ago (20),ELISpot experiments revealed that HCMV-specific T-cell responsesdirected against a broad range of antigens exist within one donor; up toeight epitopes restricted by one specific HLA-I allotype were recognizedin parallel (FIG. 9). While most of the donors showed responses to asimilar set of epitopes, some donors had highly individual patterns ofrecognition.

HCMV-Specific Memory T Cells are Multifunctional

Peptide and HLA specificity of memory T cells was tested by HLA tetramerstaining after 12 day amplification in vitro (Table 1 and FIG. 4a ). Theinventors were able to show distinct HCMV-specific CD8+ T-cellpopulations for all but one (UL44 259-267) dominant epitopes in severalPBMC samples (Table 2). Specific T-cell populations ranged from 0.3% to52% for one specificity. Functional activity of memory T cells afterstimulation with HCMV peptides could be demonstrated by ICS viadetection of IFNγ and TNF (FIG. 4b , Table 1). Predicted HLA restrictioncould be confirmed for 26 of 27 dominant epitopes. Stimulation with UL44259-267 resulted in a T-cell response mediated by CD4+ cells. Also, theinventors could demonstrate that some epitopes elicit T-cell responsesrestricted to more than one HLA-I allotype. UL46 76-84 was able toactivate CD4+ and CD8+ T cells in different PBMC samples. T-cellresponses to UL26 61-69 were detected in seven B*08+/B*51− andB*08-/B*51+ samples and were mediated by CD8+ T cells in all testeddonors. Tetramer stainings demonstrated B*08 and B*51 restriction of theepitope. However, mismatch ELISpots with B*08-/B*51− PBMC samples alsoshowed responses indicating a binding to yet more HLA allotypes whichwill have to be further investigated. In summary, the inventors wereable to further characterize dominant HCMV-derived epitopes using ICS,tetramer staining and mismatch experiments. HCMV-specific CD8+ T-cellclones effectively kill peptide-loaded or infected target cells Forexamination of cytotoxic activity CD8+ T-cell clones specific for UL2322-30 (B*07) were generated. Specificity and activity of the clones wereassessed by HLA tetramer staining and ICS. T-cell clones used forcytotoxicity experiments were highly specific and showed secretion ofIFNγ, TNF and the degranulation marker CD107a (FIG. 5a ). For furthercytotoxicity analysis the inventors applied the XCelligence system.Without reactive CD8+ T cells the HCMV-infected MRC-5 cells displayed aspecific cell index pattern as the infection proceeded and changed thecell morphology. A few hours after infection MRC-5 cells started toround up and lose adherence in comparison to uninfected cells. This isdetected by a lower cell index in the xCELLigence system. Around 20h.p.i., cell indices increased again as MRC-5 cells started tore-adhere. Finally, the cell index dropped drastically 3-5 days postinfection (depending on the applied MOI) due to cell lysis. For optimalmeasurement of T-cell dependent cytotoxicity, effector T-cells wereadded approximately 48 h.p.i. This allowed the infected cells to reachhigher cell index values prior to late cell index drop due to infection.To test peptide specificity of the CD8+ T-cell clones, mock treatedMRC-5 cells were loaded with specific or unspecific peptides or infectedwith the ΔUS2-6 mutant, and effector cells were added in a 5:1 effectorto target cell (E:T) ratio. Killing of peptide loaded cells occurredvery fast and was highly specific (FIG. 5b ); within 12 h almost allUL23 22-30 loaded target cells were killed by the specific T-cell clone.Killing of ΔUS2-6-infected cells was delayed but equally efficient. Cellindex values were much higher (less cell lysis) for cells loaded with anunspecific or no peptide when co-cultured with the UL23 22-30-specificT-cell clone. An E:T ratio dependent killing of ΔUS2-6 infected MRC-5cells started a few hours after addition of effector cells (FIG. 5c ),it reached 50% after 18 h (E:T of 1:1 and higher) and after 36 h(corresponding to 84 h.p.i.) almost all infected cells were killed bythe peptide-specific CD8+ T-cell clones. Interestingly, despite theminimal expression of HLA-I/peptide complexes on the surface ofAD169VarL infected cells, a cytolytic effect was observed at higher E:Tratios, when normalized to infected cells without effector cells (FIG.5d ). In accordance to the assumption that low a mounts of HLA-I/peptidecomplexes are responsible for the slow killing of AD169VarL infectedcells, the additional loading of specific peptide led to a dramaticincrease of lysis (FIG. 5d ).

Comparison of in Silico Epitope Prediction to Mass Spectrometric HLA-ILigand Identification

To compare the inventors' approach of identifying epitopes with anestablished in silico prediction method, the inventors applied theprediction tools SYFPEITHI and NetMHCpan3.0 to the proteome of HCMV. Theinventors ranked all peptides according to their prediction score anddetermined the position of the inventors' dominant epitopes 8 withinthis dataset (Table 2). For both SYFPEITHI and NetMHC, 25 of the 26identified dominant epitopes are among the top-scoring 2% of allpredicted peptides. This is in line with the previous experience withSYFPEITHI that the top 2% of predicted peptides usually contain thenatural T-cell epitopes (37). NetMHC categorizes its predicted peptidesinto weak (affinity<500 nM, % rank<2) and strong binders (affinity<50nM, % rank<0.5). Thus, it would be necessary to test approximately 1,300(SYFPEITHI) or 2,000 (NetMHC) peptides per HLA-I allotype and lengthvariant in order to screen epitopes from the entire HCMV proteome withinthese thresholds.

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1-22. (canceled)
 23. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, and pharmaceutical acceptable salts thereof, wherein said peptide has an overall length of between 8 and 30 amino acids.
 24. The peptide or variant according to claim 23, wherein said peptide consists of an amino acid sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 100 and optionally comprising an extension of one N- and/or one C-terminal amino acid.
 25. The peptide or variant according to claim 23, wherein the amino acid sequence is selected from SEQ ID NO: 1 to 4, 24 to 29, 40, 41, 51 to 55, 67, 68, 80, 87 to 89, and 99 to
 101. 26. The peptide or variant thereof according to claim 23, wherein said peptide is modified and/or includes non-peptide bonds.
 27. The peptide or variant thereof according to claim 23, wherein said peptide is part of a fusion protein, comprising the N-terminal amino acids of the HLA-DR antigen-associated invariant chain (li).
 28. A soluble or membrane-bound antibody, that specifically binds to the peptide or variant thereof according to claim 23, and/or the peptide or variant thereof when bound to an MHC molecule.
 29. A recombinant, soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein said ligand is at least 75% identical to an amino acid sequence according to claim
 24. 30. The T cell receptor according to claim 29, wherein said T cell receptor is a soluble molecule, and optionally comprises an effector function.
 31. A nucleic acid encoding: a peptide or variant thereof according to claim 23; a soluble or membrane-bound antibody that specifically binds to the peptide or variant thereof according to claim 23 and/or a peptide or variant thereof when bound to an MHC molecule; or a recombinant, soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein said ligand is at least 75% identical to an amino acid sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 100, and optionally comprising an extension of one N- and/or one C-terminal amino acid; wherein said nucleic acid is optionally linked to a heterologous promoter sequence.
 32. An expression vector expressing the nucleic acid according to claim
 31. 33. A recombinant host cell comprising: a recombinant peptide according to claim 23; a soluble or membrane-bound antibody that specifically binds to the peptide or variant thereof according to claim 23, and/or a peptide or variant thereof when bound to an MHC molecule; a recombinant, soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein said ligand is at least 75% identical to an amino acid sequence according to any of SEQ ID NO:1 to SEQ ID NO:100 and optionally comprising an extension of one N- and/or one C-terminal amino acid; a nucleic acid encoding a peptide or variant thereof according to claim 23; a soluble or membrane-bound antibody that specifically binds to the peptide or variant thereof according to claim 23; a recombinant, soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein said ligand is at least 75% identical to an amino acid sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 100 and optionally comprising an extension of one N- and/or one C-terminal amino acid; wherein said nucleic acid is optionally linked to a heterologous promoter sequence.
 34. A method for producing: a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, wherein said peptide has an overall length of between 8 and 30 amino acids; a soluble or membrane-bound antibody, that specifically binds to said peptide or variant thereof, and/or said peptide or variant thereof when bound to an MHC molecule; or a recombinant, soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein said ligand is at least 75% identical to an amino acid sequence according to any of SEQ ID NO:1 to SEQ ID NO:100 and optionally comprising an extension of one N- and/or one C-terminal amino acid; the method comprising culturing the host cell according to claim 33 that presents said peptide; or expresses said nucleic acid; and isolating said peptide or variant thereof, said antibody, or said T cell receptor from said host cell and/or its culture medium.
 35. An in vitro method for producing activated T lymphocytes, the method comprising contacting in vitro T cells with antigen loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate said T cells in an antigen specific manner, wherein said antigen is a peptide according to claim
 23. 36. An activated T lymphocyte, produced by the method according to claim 35, that selectively recognizes a cell that presents a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, wherein said peptide has an overall length of between 8 and 30 amino acids.
 37. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of: a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, and pharmaceutical acceptable salts thereof, wherein said peptide has an overall length of between 8 and 30 amino acids; a soluble or membrane-bound antibody, that specifically binds to said peptide or variant thereof, and/or said peptide or variant thereof when bound to an MHC molecule; a recombinant, soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein said ligand is at least 75% identical to an amino acid sequence according to any of SEQ ID NO:1 to SEQ ID NO:100 and optionally comprising an extension of one N- and/or one C-terminal amino acid; a nucleic acid encoding said peptide or variant; said soluble or membrane-bound antibody; or said recombinant, soluble or membrane-bound T cell receptor; wherein said nucleic acid is optionally linked to a heterologous promoter sequence; a recombinant host cell according to claim 33; or an activated T lymphocyte that selectively recognizes a cell that presents a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, wherein said peptide has an overall length of between 8 and 30 amino acids; and a pharmaceutically acceptable carrier, and optionally additional pharmaceutically acceptable excipients and/or stabilizers.
 38. A method for producing a personalized anti-viral vaccine, said method comprising: a) identifying at least one HCMV-associated peptide according to any one of SEQ ID NO: 1 to SEQ ID NO: 101 in a sample from said individual patient; b) selecting at least one peptide as identified in said sample from step a), and c) formulating the at least one peptide as selected in step b) into a personalized anti-viral vaccine.
 39. A kit comprising: a) a container comprising a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, and pharmaceutical acceptable salts thereof, wherein said peptide has an overall length of between 8 and 30 amino acids; a soluble or membrane-bound antibody, that specifically binds to said peptide or variant thereof, and/or said peptide or variant thereof when bound to an MHC molecule; a recombinant, soluble or membrane-bound T cell receptor that is reactive with an HLA ligand, wherein said ligand is at least 75% identical to an amino acid sequence according to any of SEQ ID NO:1 to SEQ ID NO:100 and optionally comprising an extension of one N- and/or one C-terminal amino acid; a nucleic acid encoding said peptide or variant; said soluble or membrane-bound antibody; or said recombinant, soluble or membrane-bound T cell receptor; wherein said nucleic acid is optionally linked to a heterologous promoter sequence; a host cell according to claim 33; or an activated T lymphocyte that selectively recognizes a cell that presents a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, wherein said peptide has an overall length of between 8 and 30 amino acids; or a vaccine as produced a method for producing a personalized anti-viral vaccine, said method comprising: a) identifying at least one HCMV-associated peptide according to any one of SEQ ID NO: 1 to SEQ ID NO: 101 in a sample from said individual patient; b) selecting at least one peptide as identified in said sample from step a), and c) formulating the at least one peptide as selected in step b) into a personalized anti-viral vaccine; in solution or in lyophilized form; b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; c) optionally, at least one additional peptide selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, d) optionally, instructions for (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation, and e) a substance or combination of substances acting as an adjuvant.
 40. The kit according to claim 39, further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, (v) a syringe, and vi) a mixing device.
 41. A method for treating HCMV infection in target cells in a patient, wherein said target cells present at least one peptide comprising an amino acid sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 100 and optionally comprising an extension of one N-and/or one C-terminal amino acid, wherein said method comprises administering to said patient an effective amount of: an activated T lymphocyte that selectively recognizes a cell that presents a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 101, and variant sequences of SEQ ID NO: 1 to SEQ ID NO: 101 that comprise one amino acid exchange and bind to molecule(s) of the major histocompatibility complex (MHC) and/or induce T cells cross-reacting with said variant peptide, wherein said peptide has an overall length of between 8 and 30 amino acids; the pharmaceutical composition according to claim 37, and/or the vaccine as produced by a method for producing a personalized anti-viral vaccine, said method comprising: a) identifying at least one HCMV-associated peptide according to any one of SEQ ID NO: 1 to SEQ ID NO: 101 in a sample from said individual patient; b) selecting at least one peptide as identified in said sample from step a), and c) formulating the at least one peptide as selected in step b) into a personalized anti-viral vaccine.
 42. The method according to claim 41, wherein said HCMV infection exhibits a co-morbidity with cancer, inflammatory diseases, hypertensive diseases, and/or pulmonary diseases. 